Microbial limit counting inspection method for polymyxin B sulfate

Abstract

The invention provides a polymyxin B sulfate microbial limit counting inspection method, which comprises the following steps: filtering a 1: 100 test solution and less than or equal to 100cfu test bacteria in a sterile sodium chloride aqueous solution by adopting a membrane filtration method, flushing with the sterile sodium chloride aqueous solution, and pasting a filter membrane on a culture medium plate to serve as a test group; referring to the method of the test group, taking the polymyxin B sulfate control group without adding the test bacteria; by referring to a method of a test group, respectively replacing the test solution with a 1mol / L magnesium chloride aqueous solution to serve as a bacteria solution control group; by referring to a method of a test group, replacing a test solution with a 1mol / L sterile magnesium chloride aqueous solution, and taking the test solution as a negative control group without adding test bacteria; the experimental group and the control group are cultured and counted, and the recovery ratio of the experimental bacteria is within the range of 0.5-2 and meets the requirements. The problem of applicability of the microbial limit counting inspection method is solved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a polymyxin B sulfate microbial limit counting inspection method. Background Art

[0002] Polymyxin B sulfate is a polypeptide antibiotic that is clinically used to fight infections caused by Gram-negative bacteria. It has a strong antibacterial effect on Pseudomonas aeruginosa and Escherichia coli in microbial limit tests. In the conventional method of microbial limit test, pH 7.0 sodium chloride-peptone buffer solution is used as the diluent, but polymyxin B sulfate is not fully dissolved in this solution, and the antibacterial effect of polymyxin B sulfate on the test bacteria cannot be effectively eliminated.

[0003] Based on this, the present invention provides a method for checking the microbial limit of polymyxin B sulfate by counting. Summary of the invention

[0004] The purpose of the present invention is to provide a polymyxin B sulfate microbial limit counting inspection method, which solves the problem of the dissolution state of polymyxin B sulfate in a solution and eliminates the antibacterial property of polymyxin B sulfate to Pseudomonas aeruginosa and Escherichia coli.

[0005] In one aspect, the present invention provides a method for checking the microbial limit of polymyxin B sulfate by counting, the steps comprising:

[0006] (1) Preparation of the test group:

[0007] Using the membrane filtration method, the total aerobic bacterial count is 1:100 of the test solution and the test bacteria with less than or equal to 100 cfu, filtered into a sterile sodium chloride aqueous solution, rinsed with a sterile sodium chloride aqueous solution, with the bacterial surface facing up, and the filter membrane is attached to a tryptic soy agar medium plate;

[0008] Using the membrane filtration method, the total number of molds and yeasts was counted by taking 1 mL of the test solution and less than or equal to 100 cfu of the test bacteria in a ratio of 1:10 and filtering it in a sterile sodium chloride aqueous solution, washing it with a sterile sodium chloride aqueous solution, and sticking the filter membrane on a Sabouraud dextrose agar medium plate;

[0009] (2) Preparation of the polymyxin B sulfate control group: refer to the method of the test group, but do not add the test bacteria as the polymyxin B sulfate control group;

[0010] (3) Preparation of bacterial solution control group: Referring to the method of the experimental group, 1 mol / L sterile magnesium chloride aqueous solution was used to replace the 1:100 test solution and the 1:10 test solution as the bacterial solution control group;

[0011] (4) Preparation of negative control group: refer to the method of test group, replace the test solution with 1 mol / L sterile magnesium chloride aqueous solution, and do not add test bacteria as negative control group;

[0012] (5) The above experimental and control groups were cultured and counted, and the recovery ratios of the test bacteria were all in the range of 0.5 to 2, which met the requirements.

[0013] Furthermore, the method for preparing the test solution includes: weighing polymyxin B sulfate, and using a diluent to dissolve the polymyxin B sulfate into a 1:10 test solution and a 1:100 test solution.

[0014] Furthermore, the content ratio of polymyxin B sulfate to the diluent in the 1:10 test solution is 10 g / 100 mL; the content ratio of polymyxin B sulfate to the diluent in the 1:100 test solution is 1 g / 100 mL.

[0015] Furthermore, the diluent includes a 1 mol / L magnesium chloride aqueous solution and / or a 1 g / 10 mL hydroxypropyl-beta-cyclodextrin aqueous solution.

[0016] Furthermore, the test bacteria include at least one of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0017] Furthermore, in step (1), different test bacteria are selected to perform the membrane filtration step as a test group.

[0018] Furthermore, the culturing step in step (5) comprises: inverting the tryptic soy agar plate containing Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis test bacteria at 30-35° C. for no more than 3 days, inverting the tryptic soy agar plate containing Candida albicans and Aspergillus niger at 30-35° C. for no more than 5 days, and inverting the Sabouraud dextrose agar plate containing Candida albicans and Aspergillus niger at 20-25° C. for no more than 5 days.

[0019] Furthermore, the concentration of the sodium chloride aqueous solution is 0.9 g / 100 mL.

[0020] Furthermore, the number of flushing times in step (1) is 5 times, and the amount of flushing liquid used is 100 mL / time.

[0021] Furthermore, the filter membrane comprises a nylon membrane, and the pore size comprises 0.4-0.5 μm.

[0022] Beneficial effects:

[0023] According to the antibacterial principle of polymyxin B sulfate on microorganisms, the present invention uses 1mol / L sterile magnesium chloride aqueous solution as a diluent, can fully dissolve polymyxin B sulfate, and can eliminate the antibacterial property of polymyxin B sulfate on the test bacteria. The filter membrane uses a nylon membrane to reduce the residue of polymyxin B sulfate on the filter membrane, thereby reducing the antibacterial property of the residual polymyxin B sulfate on the test bacteria. Using 0.9% sterile sodium chloride aqueous solution as a flushing liquid can eliminate the problem of polymyxin B sulfate precipitating and remaining on the filter membrane. By flushing 5 times, the polymyxin B sulfate remaining on the filter membrane is rinsed clean as much as possible, thereby reducing the antibacterial effect of polymyxin B sulfate on the test bacteria. Through the pre-treatment and membrane filtration treatment of the above-mentioned polymyxin B sulfate, the problem of the applicability of the microbial limit counting test method of this product is solved, ensuring that the above-mentioned method can effectively detect the contaminated microorganisms present in the sample, and ensuring that the inspection result is true and effective. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the present invention, the microbial limit test method is a method for checking the degree of microbial contamination of non-specified sterilized preparations and raw materials and auxiliary materials. The inspection items include the total number of aerobic bacteria, molds and yeasts. The microbial limit test should be carried out in a local Class A unidirectional air flow area under Class C environmental cleanliness. The entire inspection process should strictly comply with aseptic operation to prevent microbial re-contamination, and the work surface and environment should be regularly tested for floating bacteria, settling bacteria, dust particles, and surface microorganisms.

[0026]

[0027]

[0028] Example 1

[0029] The experimental tools in this embodiment include:

[0030] Filter membrane: Nylon membrane with a pore size of 0.45 μm. This filter membrane has lower adsorption than conventional MCE mixed fibers, which is beneficial to reduce the adhesion of polymyxin B sulfate solution during filtration and reduce the impact of residual samples on the growth of test bacteria.

[0031] Dilution: 1 mol / L sterile magnesium chloride aqueous solution. According to the antibacterial principle of polymyxin B sulfate on the test bacteria, the magnesium ions in the magnesium chloride aqueous solution can play a neutralizing role and reduce the inactivation inhibitory effect of polymyxin B sulfate on the test bacteria.

[0032] Flushing solution: 0.9 g / 100 mL of sterile sodium chloride aqueous solution. Polymyxin B sulfate has good solubility in this solution. Using this solution as a flushing solution can effectively improve the effect of flushing away the residual polymyxin B sulfate on the filter membrane.

[0033] Culture medium: commercially available tryptic soy agar medium, Sabouraud dextrose agar medium.

[0034] The polymyxin B sulfate microbial limit counting inspection method in this embodiment includes:

[0035] (1) Preliminary experiment:

[0036] Weigh polymyxin B sulfate, use 1 mol / L sterile magnesium chloride aqueous solution as diluent, dissolve polymyxin B sulfate into a 1:10 test solution, and then dilute it into a 1:100 test solution with 1 mol / L sterile magnesium chloride aqueous solution; wherein the content ratio of polymyxin B sulfate to the diluent in the 1:10 test solution is 10 g / 100 mL; the content ratio of polymyxin B sulfate to the diluent in the 1:100 test solution is 1 g / 100 mL;

[0037] (2) Preparation of test group:

[0038] Using the membrane filtration method, the total aerobic bacteria count was taken at a ratio of 1:100. 1 mL of the test solution and 100 cfu of the test bacteria were filtered into 100 mL of 0.9 g / 100 mL sterile sodium chloride aqueous solution, and rinsed with 0.9 g / 100 mL sterile sodium chloride aqueous solution for 5 times, 100 mL / time, with the bacteria side facing up, and the filter membrane was attached to a tryptic soy peptone agar plate; the test bacteria were any one of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger. Different test bacteria were selected one by one and the above experimental steps were repeated;

[0039] Using membrane filtration method, the total number of molds and yeasts was counted by taking 1 mL of the test solution and 100 cfu of the test bacteria (Candida albicans, Aspergillus niger) at a ratio of 1:10, filtering into 100 mL of 0.9 g / 100 mL sterile sodium chloride aqueous solution, washing with 0.9 g / 100 mL sterile sodium chloride aqueous solution 5 times, 100 mL / time, and sticking the filter membrane on a Sabouraud glucose agar medium plate; the test bacteria were Candida albicans or Aspergillus niger, and different test bacteria were selected one by one to repeat the above experimental steps;

[0040] (3) Preparation of polymyxin B sulfate control group:

[0041] The method of the experimental group was followed, but no test bacteria were added as the polymyxin B sulfate control group;

[0042] (4) Preparation of bacterial solution control group:

[0043] Referring to the method of the test group, 1 mol / L sterile magnesium chloride aqueous solution was used to replace the 1:100 test solution and the 1:10 test solution as the bacterial solution control group;

[0044] (5) Preparation of negative control group:

[0045] Referring to the method of the test group, the test solution was replaced by 1 mol / L sterile magnesium chloride aqueous solution, and no test bacteria were added as the negative control group;

[0046] (6) Cultivation:

[0047] The tryptic soy peptone agar plates containing the test bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis should be incubated upside down at 30-35℃ for no more than 3 days. The tryptic soy peptone agar plates containing Candida albicans and Aspergillus niger should be incubated upside down at 30-35℃ for no more than 5 days. The Sabouraud dextrose agar plates containing Candida albicans and Aspergillus niger should be incubated upside down at 20-25℃ for no more than 5 days.

[0048] It should be noted that the culture temperature in the actual culture process is a fluctuating value, and the culture can be achieved within the fluctuation value range. The specific set temperature is: the tryptic soy agar medium plate is inverted at 32°C for culture, and the Sabouraud dextrose agar medium plate is inverted at 22°C for culture;

[0049] Count the microorganisms and calculate the test bacteria recovery ratio according to the following formula:

[0050] Recovery ratio = (average number of colonies in the test group - average number of colonies in the polymyxin B sulfate control group) / average number of colonies in the bacterial solution control group;

[0051] (7) Result judgment criteria

[0052] The recovery ratio of the test bacteria is shown in Table 1 below:

[0053] Table 1

[0054]

[0055]

[0056] The above test results show that after the test sample is treated by the above method, all test bacteria are recovered by membrane filtration method in accordance with the requirements of 0.5 to 2, and the growth of the test bacteria is not significantly different from that of the bacterial solution control group, and the negative control has no sterile growth.

[0057] Therefore, the above method can be used to carry out the microbial limit count test of polymyxin B sulfate, and this method effectively solves the antibacterial problem of polymyxin B sulfate in the microbial limit count test.

[0058] Comparative Example 1

[0059] Referring to the steps in Example 1, a pH 7.0 sodium chloride-peptone buffer solution was used as a diluent to prepare a 1:10 test solution of polymyxin B sulfate. Polymyxin B sulfate was incompletely dissolved in a pH 7.0 sodium chloride-peptone buffer solution, and there was foam on the top layer of the solution. Some undissolved samples floated on the surface of the test solution, and a colloid was adsorbed on the wall of the container; polysorbate 80 was added to the above solution to prepare a solution containing 2% polysorbate 80 by volume, polymyxin B sulfate was dissolved, and the colloid adsorbed on the surface of the container disappeared and dissolved. There was no obvious insoluble solid in the solution, but the solution was turbid and opaque.

[0060] The 1:10 test solution prepared above was diluted with pH 7.0 sodium chloride-peptone buffer solution to 1:20, 1:50, and 1:100 test solutions. Pseudomonas aeruginosa was used as the test bacteria for the total aerobic bacteria count. 1 mL of the 1:100 test solution was taken for filtration. Candida albicans was used as the test bacteria for the total mold and yeast count. 1 mL of each of the 1:10, 1:20, and 1:50 test solutions was taken for filtration. A 0.1% peptone aqueous solution containing 0.1% polysorbate 80 was used as the rinse solution. Rinse 5 times, 100 mL / time.

[0061] It should be noted that the content ratio of polysorbate 80, peptone and water in the flushing solution is 0.1 mL: 0.1 g: 100 mL.

[0062] The test results showed that the recovery of the test bacteria was 0, and the antibacterial activity of the polymyxin B sulfate was not eliminated after membrane filtration. In addition, the above test process showed that polymyxin B sulfate had poor solubility in aqueous solution containing peptone. There may be factors such as insufficient dissolution or incomplete filtration and flushing, which reduced the antibacterial ability of the test sample treated by membrane filtration.

[0063] Referring to the steps in Example 1, 0.9 g / 100 mL of sterile sodium chloride aqueous solution was used as a diluent to dilute and dissolve polymyxin B sulfate to prepare a 1:10 polymyxin B sulfate solution, 1 mol / L sterile magnesium chloride aqueous solution and 1 g / 10 mL of sterile hydroxypropyl beta-cyclodextrin aqueous solution were used as diluents, and the 1:10 test solution was diluted to 1:100 test solution, respectively, using a membrane filtration method, using 0.9 g / 100 mL of sterile sodium chloride aqueous solution as a flushing liquid, and the filter membrane material was N66 nylon membrane (Zhejiang Tailin).

[0064] The test results are as follows:

[0065]

[0066] The above test results show that the above methods can effectively eliminate the antibacterial activity of polymyxin B sulfate against the test bacteria, and the key factor is that the introduction of the neutralizer can effectively eliminate its antibacterial activity.

[0067] Referring to the steps in Example 1, the preliminary test used 1 mol / L sterile magnesium chloride aqueous solution as a diluent, and polymyxin B sulfate was diluted and dissolved to prepare a 1:10 test solution, and diluted to 1:20, 1:50, and 1 mL of the above 1:10, 1:20, and 1:50 test solutions were taken to 100 mL of 0.9% sterile sodium chloride aqueous solution for filtration, and 0.9% sterile sodium chloride aqueous solution was used as a flushing liquid. MCE mixed fiber filter membrane (Zhejiang Tailin) ​​and N66 nylon membrane (Zhejiang Tailin) ​​were used for membrane filtration treatment, and the test results are as follows:

[0068]

[0069]

[0070] The above test results show that the filter membrane made of N66 nylon membrane has a significantly better recovery effect on the test bacteria than the MCE material due to its low adsorption properties. The antibacterial properties of polymyxin B sulfate show that it has a significantly stronger antibacterial effect on Pseudomonas aeruginosa than Candida albicans.

[0071] In summary, in the microbial limit counting test method of this product, 1 mol / L sterile magnesium chloride aqueous solution is used as the diluent to dilute and dissolve polymyxin B sulfate and reduce the dilution concentration, which can effectively reduce the antibacterial effect of polymyxin B sulfate on the test bacteria. The membrane filtration treatment method using N66 nylon membrane as the filter membrane and 0.9% sterile sodium chloride aqueous solution as the flushing liquid, flushing 5 times, 100 mL / time, can enhance the effect of eliminating antibacterial properties.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for checking the microbial limit of polymyxin B sulfate by counting, characterized in that the steps include: (1) Preparation of the test group: Using the membrane filtration method, the total aerobic bacterial count is 1:100 of the test solution and the test bacteria with less than or equal to 100 cfu, filtered into a sterile sodium chloride aqueous solution, rinsed with a sterile sodium chloride aqueous solution, with the bacterial surface facing up, and the filter membrane is attached to a tryptic soy agar medium plate; Using the membrane filtration method, the total number of molds and yeasts was counted by taking 1 mL of the test solution and less than or equal to 100 cfu of the test bacteria in a ratio of 1:10 and filtering it in a sterile sodium chloride aqueous solution, washing it with a sterile sodium chloride aqueous solution, and sticking the filter membrane on a Sabouraud dextrose agar medium plate; (2) Preparation of the polymyxin B sulfate control group: refer to the method of the test group, but do not add the test bacteria as the polymyxin B sulfate control group; (3) Preparation of bacterial solution control group: Referring to the method of the experimental group, 1 mol / L magnesium chloride aqueous solution was used to replace the 1:100 test solution and the 1:10 test solution as the bacterial solution control group; (4) Preparation of negative control group: refer to the method of test group, replace the test solution with 1 mol / L sterile magnesium chloride aqueous solution, and do not add test bacteria as negative control group; (5) The above experimental and control groups were cultured and counted, and the recovery ratios of the test bacteria were all in the range of 0.5 to 2, which met the requirements.

2. A polymyxin B sulfate microbial limit counting test method according to claim 1, characterized in that: The preparation method of the test solution comprises: weighing polymyxin B sulfate, and using a diluent to dissolve the polymyxin B sulfate into a 1:10 test solution and a 1:100 test solution.

3. A polymyxin B sulfate microbial limit counting test method according to claim 2, characterized in that: The content ratio of polymyxin B sulfate to the diluent in the 1:10 test solution is 10 g / 100 mL; the content ratio of polymyxin B sulfate to the diluent in the 1:100 test solution is 1 g / 100 mL.

4. A polymyxin B sulfate microbial limit counting test method according to claim 2, characterized in that: The diluent includes a 1 mol / L magnesium chloride aqueous solution and / or a 1 g / 10 mL hydroxypropyl-beta-cyclodextrin aqueous solution.

5. A polymyxin B sulfate microbial limit counting test method according to claim 1, characterized in that: The test bacteria include at least one of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger.

6. A polymyxin B sulfate microbial limit counting test method according to claim 5, characterized in that: In the step (1), different test bacteria are selected to perform the membrane filtration step as the test group.

7. A polymyxin B sulfate microbial limit counting test method according to claim 6, characterized in that: The culturing step in step (5) comprises: inverting the tryptic soy agar plate containing Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis test bacteria at 30-35° C. and culturing for no more than 3 days, inverting the tryptic soy agar plate containing Candida albicans and Aspergillus niger at 30-35° C. and culturing for no more than 5 days, and inverting the Sabouraud dextrose agar plate containing Candida albicans and Aspergillus niger at 20-25° C. and culturing for no more than 5 days.

8. A polymyxin B sulfate microbial limit counting test method according to claim 1, characterized in that: The concentration of the sodium chloride aqueous solution is 0.9 g / 100 mL.

9. A polymyxin B sulfate microbial limit counting test method according to claim 1, characterized in that: The number of flushing in step (1) is 5 times, and the amount of flushing liquid used is 100 mL / time.

10. A polymyxin B sulfate microbial limit counting test method according to claim 1, characterized in that: The filter membrane comprises a nylon membrane, and the pore size comprises 0.4-0.5 μm.

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