Benzalkonium chloride microbial limit counting inspection method

Abstract

The invention discloses a benzalkonium chloride microbial limit counting inspection method, which relates to the technical field of microbial inspection, and comprises the following steps: firstly, preparing a test solution; wetting the filter membrane with the flushing fluid to obtain a rinsed filter membrane; different amounts of test solutions are taken and added into the flushing fluid, and a first mixed solution and a second mixed solution are obtained respectively; respectively filtering the mixed solution I and the mixed solution II through a rinsed filter membrane to obtain a filtered filter membrane I and a filtered filter membrane II; respectively flushing the filtered filter membrane I and the filtered filter membrane II with flushing fluid, and counting and checking the total number of aerobic bacteria through the flushed filter membrane I; counting and checking the total number of mould and saccharomycetes through the flushed filter membrane II; according to the benzalkonium chloride microbial limit counting inspection method, the application range is expanded, the total flushing amount is smaller, the flushing frequency is smaller, the error possibility in the test process is reduced, and the result is real, accurate and effective.

Description

Technical Field

[0001] The present invention relates to a microbial testing technology, in particular to a benzalkonium chloride microbial limit counting inspection method. Background Art

[0002] Benzalkonium chloride is a white or light yellow powder, a white waxy solid or a yellow colloid. Its aqueous solution is neutral or weakly alkaline and produces a large amount of foam when shaken. Its main component is a mixture of dimethylbenzyl ammonium chloride. It is a cationic surfactant and a broad-spectrum fungicide.

[0003] In the benzalkonium chloride microbial limit count test, conventional diluents and flushing solutions (pH 7.0 chloride-peptone buffer, 0.1% peptone aqueous solution), as well as conventional methods such as increasing the amount of polysorbate 80 added, membrane filtration treatment, adjusting the dilution gradient of the test sample, reducing the filter membrane test sample filtration volume and filter membrane adsorption of the test sample, are all unable to effectively eliminate the antibacterial activity of the test sample (containing benzalkonium chloride) on the test bacteria. It is necessary to develop a new test sample treatment method to eliminate the antibacterial activity of benzalkonium chloride, combined with membrane filtration treatment, for quality inspection of microbial limits. Summary of the Invention

[0004] The object of the present invention is to provide a benzalkonium chloride microbial limit counting inspection method to overcome the above-mentioned shortcomings in the prior art.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a benzalkonium chloride microbial limit counting inspection method, comprising the following steps:

[0006] S1. Weigh benzalkonium chloride dry powder, then add 10 times the weight of the benzalkonium chloride dry powder diluent, mix well to obtain a test mother solution, and dilute the test mother solution 20 times with the diluent to obtain a test solution;

[0007] S2, wetting the filter membrane with a flushing solution to obtain a rinsed filter membrane;

[0008] S3, take 4 ml of the test solution and add it to 100 ml of the rinsing solution to obtain mixed solution 1; take 40 ml of the test solution and add it to 50 ml of the rinsing solution to obtain mixed solution 2;

[0009] S4, filtering the mixed solution 1 and the mixed solution 2 through the rinsed filter membranes, respectively, to obtain filtered filter membrane 1 and filtered filter membrane 2 after filtration; rinsing the filtered filter membrane 1 and filtered filter membrane 2 with a rinse solution 5 times, 100 ml of the rinse solution each time, to obtain rinsed filter membrane 1 and rinsed filter membrane 2;

[0010] S5. Place the rinsed filter membrane with the bacterial side facing up on a tryptic soy agar plate, invert it and incubate it at 30-35°C for 3-5 days. After the incubation is complete, count the total aerobic bacteria.

[0011] S6. Place the rinsed filter membrane with the bacterial side facing up on a Sabouraud dextrose agar plate, invert it and incubate it at 20-25°C for 5-7 days. After the incubation is complete, count and check the total number of molds and yeasts.

[0012] Furthermore, the diluent in S1 is a 40% (m / v) hydroxypropyl-beta-cyclodextrin aqueous solution.

[0013] Furthermore, the flushing solution in S2 is a 0.9% (m / v) sterile sodium chloride solution containing 1% (m / v) polysorbate 80.

[0014] Furthermore, the filter membrane in S2 is a nylon membrane with a pore size of 0.45 μm and a diameter of 47 mm.

[0015] Compared with the prior art, the benzalkonium chloride microbial limit enumeration test method provided by the present invention can be applied to benzalkonium chloride with stronger antibacterial properties, thus expanding the scope of application; the total amount of flushing is reduced, the number of flushing times is reduced, the possibility of error during the test is reduced, and the risk of affecting the integrity of the filter membrane and the biological activity of microorganisms is reduced;

[0016] This method can effectively eliminate the inhibitory effect of benzalkonium chloride on contaminating bacteria during microbial limit testing. This method can effectively detect contaminating bacteria in the test sample, ensuring that the results of microbial quality inspection are true, accurate and effective, thereby providing a more reliable basis for product quality safety assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Schematic diagram of the overall process of the microbial limit counting inspection method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Example 1:

[0021] See also Figure 1A benzalkonium chloride microbial limit counting test method comprises the following steps:

[0022] S1. Weigh benzalkonium chloride dry powder, then add 10 times the weight of the benzalkonium chloride dry powder diluent, mix well to obtain a test stock solution, and dilute the test stock solution 20 times with the diluent to obtain a test solution; the diluent is a 40% (m / v) hydroxypropyl beta-cyclodextrin aqueous solution;

[0023] S2. Wetting the filter membrane with a flushing solution to obtain a rinsed filter membrane; the flushing solution is a 0.9% (m / v) sterile sodium chloride solution containing 1% (m / v) polysorbate 80; the filter membrane is a nylon membrane with a pore size of 0.45 μm and a diameter of 47 mm;

[0024] S3, take 4 ml of the test solution and add it to 100 ml of the rinsing solution to obtain mixed solution 1; take 40 ml of the test solution and add it to 50 ml of the rinsing solution to obtain mixed solution 2;

[0025] S4, filtering the mixed solution 1 and the mixed solution 2 through the rinsed filter membranes, respectively, to obtain filtered filter membrane 1 and filtered filter membrane 2 after filtration; rinsing the filtered filter membrane 1 and filtered filter membrane 2 with a rinse solution 5 times, each time with 100 ml of the rinse solution, to obtain rinsed filter membrane 1 and rinsed filter membrane 2;

[0026] S5. Place the rinsed filter membrane with the bacterial side facing up on a tryptic soy agar plate, invert it and incubate it at 30-35°C for 3-5 days. After the incubation is complete, count the total aerobic bacteria.

[0027] S6. Place the rinsed filter membrane with the bacterial side facing up on a Sabouraud dextrose agar plate, invert it and incubate it at 20-25°C for 5-7 days. After the incubation is complete, count and check the total number of molds and yeasts.

[0028] The specific implementation method is that when conducting a microbial limit test, the operation for the test group is as follows: First, select a 40% (m / v) hydroxypropyl beta-cyclodextrin aqueous solution as a diluent, accurately weigh an appropriate amount of the test sample, add the diluent thereto, mix thoroughly, and prepare a test mother solution with a ratio of 1:10. Subsequently, the test mother solution is further diluted to obtain a 1:200 test solution for subsequent microbial count analysis. At the same time, in order to ensure the reliability of the experimental results, a negative control group is set up. In the negative control group, the diluent is used instead of the test mother solution, and the treatment is carried out according to the same operating procedure as the test group to eliminate the possible interference of other factors on the experimental results.

[0029] For microbial counts, a 0.9% (m / v) sterile sodium chloride solution containing 1% (m / v) polysorbate 80 was used as the rinse solution. This solution moistened the filter membrane, creating favorable conditions for subsequent filtration. For total aerobic bacterial counts, the following procedure was performed: 4 ml of the 1:200 test solution was transferred to a container containing 100 ml of a 0.9% sterile sodium chloride solution containing 1% polysorbate 80. Filtration was then performed using the rinsed filter membrane. After the initial filtration, the filter membrane was rinsed five times with 100 ml of the rinse solution each time to ensure that all microorganisms on the filter membrane were fully removed. To ensure the accuracy of the experimental results, two parallel culture dishes were prepared. After the filtration and rinsing is completed, gently stick the filter membrane with the bacterial side facing up on a tryptic soy agar medium plate, then place the plate upside down in a constant temperature incubator and culture it at a temperature of 30-35°C for 3-5 days. Finally, accurately count the total number of aerobic bacteria.

[0030] For counting total mold and yeast counts, the procedure is slightly different: 40 ml of the 1:200 test solution is added to 50 ml of a 0.9% sterile sodium chloride solution containing 1% polysorbate 80. Filter through a rinsed filter. Rinse the filter five times with 100 ml of the rinse solution, preparing two parallel culture dishes. After filtration and rinsing, place the filter, bacterial side up, on a Sabouraud dextrose agar plate and incubate it upside down at 20–25°C for 5–7 days. The total mold and yeast counts are then performed.

[0031] This microbial limit test method has a certain degree of sensitivity, with a detection sensitivity of ≥50 cfu / g for the total aerobic bacteria count and ≥5 cfu / g for the total mold and yeast count. However, it is worth noting that this method has certain limitations. When the microbial count standards are <50 cfu / g for the total aerobic bacteria count and <5 cfu / g for the total mold and yeast count, this method is no longer applicable.

[0032] Example 2:

[0033] This embodiment provides a technical solution based on the first embodiment: a test to verify the applicability of the benzalkonium chloride microbial limit count test method:

[0034] In this experiment, the following four different groups were set up to comprehensively evaluate the applicability of the benzalkonium chloride microbial limit enumeration test method.

[0035] Preparation of bacterial suspension: Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis are inoculated into tryptic soy broth at 30-35°C for 18-24 hours, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Candida albicans is inoculated into Sabouraud dextrose broth at 20-25°C for 2-3 days, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Aspergillus niger is inoculated into Sabouraud dextrose agar at 20-25°C for 5-7 days, spores are eluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, and diluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80 to a bacterial suspension of appropriate concentration.

[0036] Test group: Benzalkonium chloride powder was weighed, and then a diluent (10 times the weight of the benzalkonium chloride powder) was added, and the mixture was evenly mixed to obtain a test stock solution. The test stock solution was diluted 20-fold with the diluent to obtain a test solution. The filter membrane was moistened with a flushing solution to obtain a rinsed filter membrane. 4 ml of the test solution and no more than 100 cfu of test bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger suspensions) were added to 100 ml of a 0.9% sterile sodium chloride solution containing 1% polysorbate 80 to obtain an aerobic bacteria test solution. 40 ml of the test solution and no more than 100 cfu of test bacteria (Candida albicans and Aspergillus niger suspensions) were added to 50 ml of a 0.9% sterile sodium chloride solution containing 1% polysorbate 80 to obtain a mold and yeast test solution.

[0037] Bacteria solution control group: Sterile purified water was used instead of the test solution, and other operations were the same as those of the experimental group.

[0038] Test sample control group: no test bacteria were added, and other operations were the same as those of the test group.

[0039] Negative control group: The test solution was replaced by diluent, no test bacteria were added, and other operations were the same as those of the test group.

[0040] The aerobic bacteria test solutions of the four groups were filtered through the rinsed filter membranes, respectively, to obtain filtered filter membrane 1 after filtration; the filtered filter membrane 1 was rinsed 5 times with 100 ml of rinsing solution each time, to obtain rinsed filter membrane 1; the rinsed filter membrane 1 was affixed with the bacterial side upward on a tryptic soy agar medium plate, inverted and incubated at 30-35°C for 3-5 days, and the total number of aerobic bacteria was counted and checked after the incubation.

[0041] The mold and yeast test solutions of the four groups were filtered through the rinsed filter membranes respectively, and after the filtration was completed, filtered filter membrane 2 was obtained; the filtered filter membrane 2 was rinsed with a rinse solution 5 times, each time with 100 ml of the rinse solution, to obtain rinsed filter membrane 2; the rinsed filter membrane 2 was affixed with the bacterial side upward on a Sabouraud dextrose agar medium plate, inverted at 20-25°C, and cultured for 5-7 days. After the completion of the culture, the total number of molds and yeasts was counted and checked.

[0042] Acceptable standard: The recovery ratio should be between 0.5 and 2. The recovery ratio is calculated as follows:

[0043]

[0044] The results of the applicability test of the benzalkonium chloride microbial enumeration method are as follows:

[0045]

[0046] The above test results show that the recovery of test bacteria is between 0.5 and 2, which is in compliance with the regulations. The above method can effectively eliminate the antibacterial effect of the test sample on the test bacteria and can be used for the microbial limit test of benzalkonium chloride.

[0047] Example 3:

[0048] This embodiment provides a technical solution based on the first embodiment: testing the effect of a 40% (m / v) hydroxypropyl-beta-cyclodextrin aqueous solution on microorganisms.

[0049] In this experiment, the following four different groups were set up to comprehensively evaluate the effects of 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution on different microorganisms.

[0050] Preparation of bacterial suspension: Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis are inoculated into tryptic soy broth at 30-35°C for 18-24 hours, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Candida albicans is inoculated into Sabouraud dextrose broth at 20-25°C for 2-3 days, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Aspergillus niger is inoculated into Sabouraud dextrose agar at 20-25°C for 5-7 days, spores are eluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, and diluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80 to a bacterial suspension of appropriate concentration.

[0051] Test Group: Accurately measure 25 ml of an aqueous solution containing 40% (m / v) hydroxypropyl-β-cyclodextrin and divide evenly into five sterile test tubes that have been rigorously sterilized, with each tube receiving 5 ml of the solution. Next, add no more than 50 μl of a suspension of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger to each of these five test tubes. The test tubes containing these bacterial suspensions serve as the bacterial test solutions, ensuring that each ml of the test solution contains no more than 100 cfu.

[0052] Bacterial solution control group: To eliminate the interference of the 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution on the experimental results, a bacterial solution control group was established. Specifically, purified water was used to replace the 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution in the experimental group. All other steps were identical to those for the experimental group. Specifically, 25 ml of purified water was dispensed into five sterile test tubes, 5 ml each. The same bacterial suspension of the same type and amount (no more than 50 μl) as in the experimental group was then added to each tube to prepare a bacterial test solution. The bacterial count per ml of the bacterial test solution was ensured to be no more than 100 cfu. The bacterial suspension control group serves as a benchmark for comparing the effects of the hydroxypropyl-β-cyclodextrin aqueous solution in the experimental group on microbial growth.

[0053] Test sample group: Inject an appropriate amount of a 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution into a sterile plate. Do not add any test bacteria. Follow the same procedures as for the experimental group. Follow subsequent culture and count procedures to determine the microbial status of the test sample at the beginning of the experiment, providing important evidence for subsequent analysis.

[0054] Negative control group: Serving as a blank control for the entire experiment, the negative control group did not contain the 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution or the bacterial solution, but underwent the same pretreatment procedures as the experimental group. This step was to eliminate any interference from factors other than the bacterial solution and the test sample during the experiment, thereby ensuring the accuracy of the experimental results.

[0055] For the total aerobic bacterial count, 1 ml of solution was taken from each of the four groups and injected into a sterile plate. 15 to 20 ml of melted trypticase soy agar medium at a temperature not exceeding 45°C was then injected into the plate. The temperature was controlled within this range to prevent damage to the microorganisms caused by high temperatures and to ensure that the microorganisms were fully mixed with the culture medium at an appropriate temperature. Two plates were prepared in parallel to increase the credibility of the experimental results. The plates were inverted and incubated in an incubator at 30 to 35°C for 3 to 5 days. This temperature range is the ideal temperature range for the growth of aerobic bacteria. After a 3 to 5-day incubation period, aerobic bacteria can fully grow and reproduce, facilitating subsequent counting and inspection.

[0056] For the count of the total number of molds and yeasts, 1 ml of solution was taken from each of the four groups and injected into a sterile plate, followed by 15 to 20 ml of Sabouraud dextrose agar melted at a temperature not exceeding 45°C. Sabouraud dextrose agar is a culture medium specifically used for the cultivation of molds and yeasts, and its composition can meet the growth requirements of these microorganisms. Similarly, two plates were prepared in parallel and inverted in an incubator at 20 to 25°C for 5 to 7 days. 20 to 25°C is the temperature range suitable for the growth of molds and yeasts. The 5 to 7 days of incubation can ensure that these relatively slow-growing microorganisms fully develop and form countable colonies.

[0057] Throughout the experimental process, each step required strict adherence to aseptic operation principles to ensure a clean experimental environment and prevent contamination by external microorganisms. By culturing and counting each group, the effects of a 40% (m / v) hydroxypropyl-β-cyclodextrin aqueous solution on different microorganisms could be accurately assessed.

[0058] The test results of the effect of 40% (m / v) hydroxypropyl beta-cyclodextrin aqueous solution on microorganisms are as follows:

[0059]

[0060]

[0061] Acceptable standard: The recovery ratio should be between 0.5 and 2. The recovery ratio calculation formula is as follows:

[0062]

[0063] The above test results show that the aqueous solution containing 40% (m / v) hydroxypropyl-beta-cyclodextrin has no effect on the growth of the above test bacteria, and the test bacteria recovery ratio is between 0.5 and 2, which meets the requirements. The solution can be used for microbial limit testing.

[0064] Example 4:

[0065] This embodiment provides a technical solution based on the first embodiment: a test on the effect of 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 on microorganisms.

[0066] In this experiment, the following four different groups were set up to comprehensively evaluate the effects of 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 on different microorganisms.

[0067] Preparation of bacterial suspension: Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis are inoculated into tryptic soy broth at 30-35°C for 18-24 hours, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Candida albicans is inoculated into Sabouraud dextrose broth at 20-25°C for 2-3 days, and diluted with 0.9% sterile sodium chloride solution to a bacterial suspension of appropriate concentration; Aspergillus niger is inoculated into Sabouraud dextrose agar at 20-25°C for 5-7 days, spores are eluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, and diluted with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80 to a bacterial suspension of appropriate concentration.

[0068] Test Group: Accurately measure 25 ml of a 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 and divide evenly into five sterile test tubes that have been rigorously sterilized, with each tube receiving 5 ml of solution. Next, add no more than 50 μl of a suspension of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger to each of these five test tubes. The test tubes containing these bacterial suspensions serve as the bacterial test solutions, ensuring that each ml of the test solution contains no more than 100 cfu.

[0069] Bacterial solution control group: To eliminate the potential interference of polysorbate 80 added to the 0.9% sterile sodium chloride solution on the experimental results, a bacterial solution control group was established. Specifically, the 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 in the experimental group was replaced with 0.9% sterile sodium chloride solution. All other procedures were identical to those for the experimental group. Specifically, 25 ml of purified water was dispensed into five sterile test tubes, 5 ml each. The same bacterial suspension of the same type and volume (no more than 50 μl) as in the experimental group was then added to each tube to prepare a bacterial test solution. The bacterial count per ml of the bacterial test solution was maintained at no more than 100 cfu. The bacterial solution control group served as a baseline for comparison with the effect of the 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 in the experimental group on microbial growth.

[0070] Test sample group: Inject an appropriate amount of 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 into a sterile dish. Do not add any test bacteria. Follow the same procedures as for the experimental group. Follow subsequent culture and enumeration procedures to determine the microbial status of the test sample at the beginning of the experiment, providing important evidence for subsequent analysis of the results.

[0071] Negative control group: Serving as a blank control for the entire experiment, the negative control group undergoes the same pretreatment procedures as the experimental group, except for the addition of 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 and the bacterial solution. This step is intended to eliminate interference from factors other than the bacterial solution and the test sample during the experimental process, thereby ensuring the accuracy of the experimental results.

[0072] For the total aerobic bacterial count, 1 ml of solution was taken from each of the four groups and injected into a sterile plate. 15 to 20 ml of melted trypticase soy agar medium at a temperature not exceeding 45°C was then injected into the plate. The temperature was controlled within this range to prevent damage to the microorganisms caused by high temperatures and to ensure that the microorganisms were fully mixed with the culture medium at an appropriate temperature. Two plates were prepared in parallel to increase the credibility of the experimental results. The plates were inverted and incubated in an incubator at 30 to 35°C for 3 to 5 days. This temperature range is the ideal temperature range for the growth of aerobic bacteria. After a 3 to 5-day incubation period, aerobic bacteria can fully grow and reproduce, facilitating subsequent counting and inspection.

[0073] For the count of the total number of molds and yeasts, 1 ml of solution was taken from each of the four groups and injected into a sterile plate, followed by 15 to 20 ml of Sabouraud dextrose agar melted at a temperature not exceeding 45°C. Sabouraud dextrose agar is a culture medium specifically used for the cultivation of molds and yeasts, and its composition can meet the growth requirements of these microorganisms. Similarly, two plates were prepared in parallel and inverted in an incubator at 20 to 25°C for 5 to 7 days. 20 to 25°C is the temperature range suitable for the growth of molds and yeasts. The 5 to 7 days of incubation can ensure that these relatively slow-growing microorganisms fully develop and form countable colonies.

[0074] Throughout the experimental process, each step required strict adherence to aseptic techniques to ensure a clean experimental environment and prevent contamination by external microorganisms. By culturing and counting each group, we were able to accurately assess the effects of a 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 on different microorganisms.

[0075] Acceptable standard: The recovery ratio should be between 0.5 and 2. The recovery ratio is calculated as follows:

[0076]

[0077] The test results of the effect of 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 on microorganisms are as follows:

[0078]

[0079] Conclusion: The above test results show that 0.9% sterile sodium chloride solution containing 1% (m / v) polysorbate 80 has no effect on the growth of the above test bacteria, and the test bacteria recovery ratio is between 0.5 and 2, which is in line with the regulations. This solution can be used for microbial limit tests.

[0080] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A benzalkonium chloride microbial limit enumeration test method, characterized in that, The steps include: S1. Weigh benzalkonium chloride dry powder, then add 10 times the weight of the benzalkonium chloride dry powder diluent, mix well to obtain a test mother solution, and dilute the test mother solution 20 times with the diluent to obtain a test solution; S2, wetting the filter membrane with a flushing solution to obtain a rinsed filter membrane; S3, take 4 ml of the test solution and add it to 100 ml of the rinsing solution to obtain mixed solution 1; take 40 ml of the test solution and add it to 50 ml of the rinsing solution to obtain mixed solution 2; S4, filtering the mixed solution 1 and the mixed solution 2 through the rinsed filter membranes, respectively, to obtain filtered filter membrane 1 and filtered filter membrane 2 after filtration; rinsing the filtered filter membrane 1 and filtered filter membrane 2 with a rinse solution 5 times, 100 ml of the rinse solution each time, to obtain rinsed filter membrane 1 and rinsed filter membrane 2; S5. Place the rinsed filter membrane with the bacterial side facing up on a tryptic soy agar plate, invert it and incubate it at 30-35°C for 3-5 days. After the incubation is complete, count the total aerobic bacteria. S6. Place the rinsed filter membrane with the bacterial side facing up on a Sabouraud dextrose agar plate, invert it and incubate it at 20-25°C for 5-7 days. After the incubation is complete, count and check the total number of molds and yeasts.

2. a kind of benzalkonium chloride microbial limit counting inspection method according to claim 1, is characterized in that, The diluent S1 is a 40% (m / v) hydroxypropyl-beta-cyclodextrin aqueous solution.

3. a kind of benzalkonium chloride microbial limit counting inspection method according to claim 1, is characterized in that, The flushing solution in S2 is a 0.9% (m / v) sterile sodium chloride solution containing 1% (m / v) polysorbate 80.

4. a kind of benzalkonium chloride microbial limit counting inspection method according to claim 1, is characterized in that, The filter membrane S2 is a nylon membrane with a pore size of 0.45 μm and a diameter of 47 mm.

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