Microbial limit test method of nifuratel nystatin vaginal soft capsule

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a microbial limit detection method of nifuratel nystatin vaginal soft capsules, which comprises the following specific steps: preparing bacterial liquid: preparing a suspension A, a suspension B and a suspension C; preparing a test solution, namely putting 10g of nifuratel nystatin vaginal soft capsule into a container, then adding 100mL of sterile sodium chloride-peptone buffer solution into the container, shaking up, then adding 10mL of suspension A, centrifuging, taking a clear layer as the test solution A of 1: 10 after centrifuging, and taking the clear layer as the test solution B of 1: 10; the nifuratel nystatin vaginal soft capsule detection device has the advantages that the nifuratel nystatin vaginal soft capsule can be subjected to microbial limit detection in various environments, and the nifuratel nystatin vaginal soft capsule can be subjected to microbial limit detection in various environments; further, the accuracy of microbial limit examination on the nifuratel nystatin vaginal soft capsule can be improved to a certain extent.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, in particular to a microbial limit inspection method for nifuratel nystatin vaginal soft capsules. Background Art

[0002] Symptoms of bacterial vaginosis include increased vaginal discharge with a fishy odor, which worsens after sexual intercourse and may be accompanied by vulvar itching or burning sensation. However, 10% to 40% of patients have no symptoms and are only discovered during physical examinations.

[0003] The symptoms of candidal vaginitis are vulvar itching, vulvar and vaginal burning pain, increased leucorrhea in the form of tofu dregs, sometimes accompanied by frequent urination, dysuria, and pain during sexual intercourse. During gynecological examination, a white film can be seen attached to the inner side of the labia minora and the vaginal mucosa, which can be wiped off to reveal a red and swollen mucosal surface. In the acute phase, a damaged erosive surface can be seen.

[0004] Nifuratel Nystatin Vaginal Soft Capsules are indicated for bacterial vaginosis, Trichomonas vaginitis, Candida vulvovaginitis, and mixed vaginal infections. Nifuratel is a nitrofuran derivative with a broad-spectrum antimicrobial effect, and is active against Trichomonas, bacteria, and Candida albicans.

[0005] After the production of the current nifuratel nystatin vaginal soft capsule, a microbial limit test is usually performed on it, but currently only one environment is used to test the microbial limit, which may result in low test accuracy. Therefore, a method for testing the microbial limit of nifuratel nystatin vaginal soft capsule is invented. Summary of the invention

[0006] In view of the above problems and / or the problems existing in the existing microbial limit test method for nifuratel nystatin vaginal soft capsules, the present invention is proposed.

[0007] Therefore, the object of the present invention is to provide a microbial limit test method for nifuratel nystatin vaginal soft capsules, which can solve the above-mentioned existing problems.

[0008] To solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions:

[0009] A method for microbial limit test of nifuratel nystatin vaginal soft capsules comprises the following specific steps:

[0010] S1, bacterial suspension preparation:

[0011] preparing suspension A, suspension B and suspension C;

[0012] S2, test solution preparation:

[0013] 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension A was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution A;

[0014] 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension B was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution B;

[0015] Place 10 g of nifuratel nystatin vaginal soft capsules in a container, then add 100 mL of sterile sodium chloride-peptone buffer to the container, shake it well, then add 10 mL of suspension C, and centrifuge. After centrifugation, take the clear layer as 1:10 test solution C;

[0016] S3, Determination of the total number of aerobic bacteria, bacteria, molds and yeasts:

[0017] S31, experimental group A: inject 1mL of 1:10 test solution A into the membrane filter for microbial limit, and rinse it with 500mL of sterile sodium chloride-peptone buffer for 5 times. In the last rinse, add suspension A to the sterile sodium chloride-peptone buffer. Then, after filtering, remove the filter membrane and stick the bacteria upward on the nutrient agar medium plate. Then, place it in an environment of 30-40℃ for 24-48h. Then, stick Candida albicans and Aspergillus niger face up on the rose bengal sodium agar medium plate, and place it in an environment of 25-30℃ for 24-72h. During this process, the results need to be observed daily and the number of colonies should be counted.

[0018] Experimental group B: 1 mL of 1:10 test solution B was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension B was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted.

[0019] Experimental group C: 1 mL of 1:10 test solution C was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension C was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted.

[0020] S32, bacterial solution A group: repeat the experimental group A, and replace the test solution A with 1 mL of sterile sodium chloride-peptone buffer;

[0021] Bacterial solution group B: Repeat the experimental group B, and replace the test solution B with 1 mL of sterile sodium chloride-peptone buffer;

[0022] Bacterial solution group C: Repeat the experimental group C, and replace the test solution C with 1 mL of sterile sodium chloride-peptone buffer;

[0023] S33, test sample control group A: repeat experimental group A, replacing suspension A with sterile sodium chloride-peptone buffer;

[0024] Test sample control group B: Repeat experimental group B, replacing suspension B with sterile sodium chloride-peptone buffer;

[0025] Test sample control group C: Repeat experimental group C, replacing suspension C with sterile sodium chloride-peptone buffer;

[0026] S34, determination of the total number of aerobic bacteria, bacteria, molds and yeasts:

[0027] The ratio of the colony count of the test group minus the colony count of the test sample group to the colony count of the bacterial solution group is within the range of 0.5 to 2, which is qualified.

[0028] S4, Inspection of control bacteria:

[0029] S41, inspection method:

[0030] Test group A: 10 mL of 1:10 test solution A was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0031] Test group B: 10 mL of 1:10 test solution B was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0032] Test group C: 10 mL of 1:10 test solution C was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0033] Negative control group: replace the 1:10 test solution in the test sample group with sterile sodium chloride-peptone buffer.

[0034] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules of the present invention, the temperature of the sterile sodium chloride-peptone buffer solution is 45° C., the sterile sodium chloride-peptone buffer solution contains 1% polysorbate 80, and the pH value of the sterile sodium chloride-peptone buffer solution is 7.0.

[0035] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules of the present invention, the suspension A is a Staphylococcus aureus suspension, a Pseudomonas aeruginosa suspension, a Bacillus subtilis suspension, a Candida albicans suspension, an Aspergillus niger spore suspension and an Escherichia coli suspension.

[0036] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules described in the present invention, the suspension B is an anaerobic bacteria suspension, a Staphylococcus aureus suspension, a Pseudomonas aeruginosa suspension, a Bacillus subtilis suspension, a Candida albicans suspension, an Aspergillus niger spore suspension and an Escherichia coli suspension.

[0037] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules described in the present invention, the suspension C is a penicillin suspension, a Staphylococcus aureus suspension, a Pseudomonas aeruginosa suspension, a Bacillus subtilis suspension, a Candida albicans suspension, an Aspergillus niger spore suspension and an Escherichia coli suspension.

[0038] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules of the present invention, the preparation method of the Staphylococcus aureus suspension is to take a fresh culture of Staphylococcus aureus cultured at 30-40°C for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL.

[0039] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules described in the present invention, the preparation method of the Pseudomonas aeruginosa suspension is to take a fresh culture of Pseudomonas aeruginosa cultured at 30-40°C for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL.

[0040] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules of the present invention, the preparation method of the Bacillus subtilis suspension is to take a fresh culture of Bacillus subtilis cultured at 30-40°C for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL.

[0041] As a preferred embodiment of the microbial limit test method for nifuratel nystatin vaginal soft capsules of the present invention, the preparation method of the Candida albicans suspension is as follows: a fresh culture of Candida albicans cultured at 20-30°C for 2-3 days is taken, and a bacterial suspension containing 60-100 cfu per 1 mL is prepared using 0.9% sterile sodium chloride solution.

[0042] As a preferred embodiment of the microbial limit test method for the nifuratel nystatin vaginal soft capsule of the present invention, the preparation method of the Aspergillus niger spore suspension is as follows: a fresh culture of Aspergillus niger cultured at 20-30° C. for 5-6 days is taken, 3-4 mL of a 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80 is added, the spores are eluted, the spore suspension is sucked out into a sterile test tube, and a spore suspension containing 60-100 cfu per 1 mL is prepared using a 0.9% sterile sodium chloride solution.

[0043] Compared with existing technologies:

[0044] The present invention has the capability of performing microbial limit tests on the nifuratel-nystatin vaginal soft capsules under various environments, and can improve the accuracy of the microbial limit tests on the nifuratel-nystatin vaginal soft capsules to a certain extent by performing microbial limit tests on the nifuratel-nystatin vaginal soft capsules under various environments. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0046] The present invention provides a method for microbial limit inspection of nifuratel nystatin vaginal soft capsules, comprising the following specific steps:

[0047] S1, bacterial suspension preparation:

[0048] preparing suspension A, suspension B and suspension C;

[0049] Suspension A is a suspension of Staphylococcus aureus, a suspension of Pseudomonas aeruginosa, a suspension of Bacillus subtilis, a suspension of Candida albicans, a suspension of Aspergillus niger spores, and a suspension of Escherichia coli;

[0050] Suspension B is anaerobic bacteria suspension, Staphylococcus aureus suspension, Pseudomonas aeruginosa suspension, Bacillus subtilis suspension, Candida albicans suspension, Aspergillus niger spore suspension and Escherichia coli suspension;

[0051] Suspension C is penicillin suspension, Staphylococcus aureus suspension, Pseudomonas aeruginosa suspension, Bacillus subtilis suspension, Candida albicans suspension, Aspergillus niger spore suspension and Escherichia coli suspension;

[0052] The preparation method of the Staphylococcus aureus suspension is to take a fresh culture of Staphylococcus aureus cultured at 30-40° C. for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL;

[0053] The preparation method of the Pseudomonas aeruginosa suspension is to take a fresh culture of Pseudomonas aeruginosa cultured at 30-40°C for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL;

[0054] The preparation method of the Bacillus subtilis suspension is to take a fresh culture of Bacillus subtilis cultured at 30-40°C for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL;

[0055] The preparation method of the Candida albicans suspension is to take a fresh culture of Candida albicans cultured at 20-30°C for 2-3 days, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL;

[0056] The preparation method of the Aspergillus niger spore suspension is as follows: taking a fresh culture of Aspergillus niger cultured at 20-30° C. for 5-6 days, adding 3-4 mL of a 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, eluting the spores, sucking out the spore suspension into a sterile test tube, and using the 0.9% sterile sodium chloride solution to prepare a spore suspension containing 60-100 cfu per 1 mL;

[0057] S2, test solution preparation:

[0058] 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension A was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution A;

[0059] 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension B was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution B;

[0060] Place 10 g of nifuratel nystatin vaginal soft capsules in a container, then add 100 mL of sterile sodium chloride-peptone buffer to the container, shake it well, then add 10 mL of suspension C, and centrifuge. After centrifugation, take the clear layer as 1:10 test solution C;

[0061] S3, Determination of the total number of aerobic bacteria, bacteria, molds and yeasts:

[0062] S31, experimental group A: inject 1mL of 1:10 test solution A into the membrane filter for microbial limit, and rinse it with 500mL of sterile sodium chloride-peptone buffer for 5 times. In the last rinse, add suspension A to the sterile sodium chloride-peptone buffer. Then, after filtering, remove the filter membrane and stick the bacteria upward on the nutrient agar medium plate. Then, place it in an environment of 30-40℃ for 24-48h. Then, stick Candida albicans and Aspergillus niger face up on the rose bengal sodium agar medium plate, and place it in an environment of 25-30℃ for 24-72h. During this process, the results need to be observed daily and the number of colonies should be counted.

[0063] Experimental group B: 1 mL of 1:10 test solution B was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension B was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted.

[0064] Experimental group C: 1 mL of 1:10 test solution C was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension C was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted.

[0065] S32, bacterial solution A group: repeat the experimental group A, and replace the test solution A with 1 mL of sterile sodium chloride-peptone buffer;

[0066] Bacterial solution group B: Repeat the experimental group B, and replace the test solution B with 1 mL of sterile sodium chloride-peptone buffer;

[0067] Bacterial solution group C: Repeat the experimental group C, and replace the test solution C with 1 mL of sterile sodium chloride-peptone buffer;

[0068] S33, test sample control group A: repeat experimental group A, replacing suspension A with sterile sodium chloride-peptone buffer;

[0069] Test sample control group B: Repeat experimental group B, replacing suspension B with sterile sodium chloride-peptone buffer;

[0070] Test sample control group C: Repeat experimental group C, replacing suspension C with sterile sodium chloride-peptone buffer;

[0071] S34, determination of the total number of aerobic bacteria, bacteria, molds and yeasts:

[0072] The ratio of the colony count of the test group minus the colony count of the test sample group to the colony count of the bacterial solution group is within the range of 0.5 to 2, which is qualified.

[0073] S4, Inspection of control bacteria:

[0074] S41, inspection method:

[0075] Test group A: 10 mL of 1:10 test solution A was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0076] Test group B: 10 mL of 1:10 test solution B was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0077] Test group C: 10 mL of 1:10 test solution C was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours.

[0078] Negative control group: replace the 1:10 test solution in the test sample group with sterile sodium chloride-peptone buffer.

[0079] The temperature of the sterile sodium chloride-peptone buffer solution is 45° C., the sterile sodium chloride-peptone buffer solution contains 1% polysorbate 80, and the pH value of the sterile sodium chloride-peptone buffer solution is 7.0.

[0080] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for microbial limit test of nifuratel nystatin vaginal soft capsules, characterized in that: The specific steps are as follows: S1, bacterial suspension preparation: preparing suspension A, suspension B and suspension C; S2, test solution preparation: 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension A was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution A; 10 g of nifuratel nystatin vaginal soft capsules were placed in a container, and then 100 mL of sterile sodium chloride-peptone buffer was added to the container, and then the container was shaken, and then 10 mL of suspension B was added, and centrifuged. After centrifugation, the clear layer was taken as 1:10 test solution B; Place 10 g of nifuratel nystatin vaginal soft capsules in a container, then add 100 mL of sterile sodium chloride-peptone buffer to the container, shake it well, then add 10 mL of suspension C, and centrifuge. After centrifugation, take the clear layer as 1:10 test solution C; S3, Determination of the total number of aerobic bacteria, bacteria, molds and yeasts: S31, experimental group A: inject 1mL of 1:10 test solution A into the membrane filter for microbial limit, and rinse it with 500mL of sterile sodium chloride-peptone buffer for 5 times. In the last rinse, add suspension A to the sterile sodium chloride-peptone buffer. Then, after filtering, remove the filter membrane and stick the bacteria upward on the nutrient agar medium plate. Then, place it in an environment of 30-40℃ for 24-48h. Then, stick Candida albicans and Aspergillus niger face up on the rose bengal sodium agar medium plate, and place it in an environment of 25-30℃ for 24-72h. During this process, the results need to be observed daily and the number of colonies should be counted. Experimental group B: 1 mL of 1:10 test solution B was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension B was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted. Experimental group C: 1 mL of 1:10 test solution C was injected into the membrane filter for microbial limit, and rinsed with 500 mL of sterile sodium chloride-peptone buffer for 5 times. During the last rinse, suspension C was added to the sterile sodium chloride-peptone buffer. After filtration, the filter membrane was removed and the bacteria were attached to the nutrient agar medium plate with the surface facing upwards. After that, it was placed in an environment of 30-40°C for 24-48 hours. Then, Candida albicans and Aspergillus niger were attached to the rose bengal sodium agar medium plate with the surface facing upwards, and it was placed in an environment of 25-30°C for 24-72 hours. During this process, the results should be observed daily and the number of colonies should be counted. S32, bacterial solution A group: repeat the experimental group A, and replace the test solution A with 1 mL of sterile sodium chloride-peptone buffer; Bacterial solution group B: Repeat the experimental group B, and replace the test solution B with 1 mL of sterile sodium chloride-peptone buffer; Bacterial solution group C: Repeat the experimental group C, and replace the test solution C with 1 mL of sterile sodium chloride-peptone buffer; S33, test sample control group A: repeat experimental group A, replacing suspension A with sterile sodium chloride-peptone buffer; Test sample control group B: Repeat experimental group B, replacing suspension B with sterile sodium chloride-peptone buffer; Test sample control group C: Repeat experimental group C, replacing suspension C with sterile sodium chloride-peptone buffer; S34, determination of the total number of aerobic bacteria, bacteria, molds and yeasts: The ratio of the colony count of the test group minus the colony count of the test sample group to the colony count of the bacterial solution group is within the range of 0.5 to 2, which is qualified. S4, Inspection of control bacteria: S41, inspection method: Test group A: 10 mL of 1:10 test solution A was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours. Test group B: 10 mL of 1:10 test solution B was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours. Test group C: 10 mL of 1:10 test solution C was divided into 3 portions and injected into the membrane filter for microbial limit, and rinsed with sterile sodium chloride-peptone buffer for 5 times. 10-100 CFU of Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were added during each rinse. 3% lecithin was added during the first rinse, and the test bacteria were added during the last rinse. After that, 100 mL of nutrient broth medium, Sabouraud dextrose liquid medium and bile salt lactose medium were added to the membrane, shaken well, and cultured for 24-72 hours. Then, the culture was taken out and streaked on mannitol sodium chloride agar medium, Sabouraud dextrose agar plate and cetyltrimethylammonium bromide agar plate, and cultured for 24-72 hours. Negative control group: replace the 1:10 test solution in the test sample group with sterile sodium chloride-peptone buffer.

2. The microbial limit test method of a nifuratel nystatin vaginal soft capsule according to claim 1, characterized in that: The temperature of the sterile sodium chloride-peptone buffer solution is 45° C. The sterile sodium chloride-peptone buffer solution contains 1% polysorbate 80, and the pH value of the sterile sodium chloride-peptone buffer solution is 7.

0.

3. The microbial limit test method of a nifuratel nystatin vaginal soft capsule according to claim 2, characterized in that: The suspension A is a suspension of Staphylococcus aureus, a suspension of Pseudomonas aeruginosa, a suspension of Bacillus subtilis, a suspension of Candida albicans, a suspension of Aspergillus niger spores and a suspension of Escherichia coli.

4. The microbial limit test method of a nifuratel nystatin vaginal soft capsule according to claim 3, characterized in that: The suspension B is anaerobic bacteria suspension, Staphylococcus aureus suspension, Pseudomonas aeruginosa suspension, Bacillus subtilis suspension, Candida albicans suspension, Aspergillus niger spore suspension and Escherichia coli suspension.

5. The microbial limit test method of nifuratel nystatin vaginal soft capsule according to claim 4, characterized in that: The suspension C is penicillin suspension, Staphylococcus aureus suspension, Pseudomonas aeruginosa suspension, Bacillus subtilis suspension, Candida albicans suspension, Aspergillus niger spore suspension and Escherichia coli suspension.

6. The microbial limit test method for nifuratel nystatin vaginal soft capsule according to claim 5, characterized in that: The preparation method of the Staphylococcus aureus suspension is as follows: fresh Staphylococcus aureus cultured at 30-40° C. for 18-24 hours is taken, and a bacterial suspension containing 60-100 cfu per 1 mL is prepared using 0.9% sterile sodium chloride solution.

7. The microbial limit test method for nifuratel nystatin vaginal soft capsule according to claim 5, characterized in that: The preparation method of the Pseudomonas aeruginosa suspension is to take fresh culture of Pseudomonas aeruginosa cultured at 30-40° C. for 18-24 hours, and use 0.9% sterile sodium chloride solution to prepare a bacterial suspension containing 60-100 cfu per 1 mL.

8. The microbial limit test method for nifuratel nystatin vaginal soft capsule according to claim 5, characterized in that: The preparation method of the Bacillus subtilis suspension is as follows: fresh culture of Bacillus subtilis cultured at 30-40° C. for 18-24 hours is taken, and a bacterial suspension containing 60-100 cfu per 1 mL is prepared using 0.9% sterile sodium chloride solution.

9. The microbial limit test method for nifuratel nystatin vaginal soft capsule according to claim 5, characterized in that: The preparation method of the Candida albicans suspension is as follows: fresh culture of Candida albicans cultured at 20-30° C. for 2-3 days is taken, and a bacterial suspension containing 60-100 cfu per 1 mL is prepared with 0.9% sterile sodium chloride solution.

10. The microbial limit test method for nifuratel nystatin vaginal soft capsule according to claim 5, characterized in that: The preparation method of the Aspergillus niger spore suspension comprises the following steps: taking a fresh culture of Aspergillus niger cultured at 20-30° C. for 5-6 days, adding 3-4 mL of a 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, eluting spores, sucking out the spore suspension into a sterile test tube, and using the 0.9% sterile sodium chloride solution to prepare a spore suspension containing 60-100 cfu per 1 mL.