Collection system and collection method for microorganisms in medicinal gas

Abstract

The collecting system comprises a flow controller and a collector, the bottom of the flow controller is connected with a gas cylinder adapter through a pressure reducing valve, a gas pipe on the upper portion of the flow controller is connected with the collector, the collector is provided with a bottle plug, and the gas pipe extends into the bottom of the collector through the bottle plug. The tail part of the air pipe is designed to be spiral, a plurality of air holes are formed in the outer side of the spiral pipe, and the upper part of the collector is provided with an air outlet pipeline and is connected with a check valve; the collecting solution is a mixed solution of medical glycerol and 0.9% sterile sodium chloride solution in a volume ratio of 1: 1. The collection method comprises the steps of collection liquid preparation, sterilization, disinfection, gas interception, collection liquid culture and microorganism quantification. The collection system and the collection method are convenient and rapid, easy to sterilize, high in microorganism keep-alive rate, high in rejection rate and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of collection and detection of microorganisms in medicinal gas, and in particular to a system and method for collecting microorganisms in medicinal gas. Background Art

[0002] Medicinal gases play a vital role in the medical field due to their unique physical and chemical properties. Medicinal gases such as sulfur hexafluoride (SF6) have a density approximately five times that of air. This high density allows it to effectively fill the intraocular space during eye surgery, providing stable support and thus assisting retinal reattachment. Furthermore, sulfur hexafluoride can remain in the eye for an extended period, providing an ample window for retinal repair. Inert gases such as sulfur hexafluoride and octafluoropropane (C3F8) are also commonly used as contrast agents in medical imaging. Due to their low solubility and excellent stability, they can form stable bubbles in the human body, thereby enhancing image contrast. This suggests that medicinal gases, with their advantages such as non-toxicity, low solubility, and ability to remain in the human body for extended periods, will continue to expand their application in clinical medicine.

[0003] According to laws and regulations, as well as requirements for human safety, pharmaceutical gases are primarily used for injections, which place high quality standards on the market and require strict control of impurities and microbial content. Existing pharmaceutical gas microbial collection systems and solutions are primarily designed for ambient air sampling, but these systems require low accuracy and large sampling volumes, making them unsuitable for testing valuable APIs.

[0004] According to the requirements of the microbial limit test method of the "Chinese Pharmacopoeia", the number of microorganisms in drugs should be counted after sampling and culturing. Therefore, the representativeness and microbial activity of the sample should be guaranteed as much as possible. For example, when using filter membrane sampling, the dryness and dehydration of the filter membrane environment and the static electricity generated can easily cause the death of microorganisms. Subsequent elution will also affect the activity of microorganisms. In addition, the filter membrane is prone to uneven clogging, making it difficult to maintain stable sampling and resulting in inaccurate culture results. If the direct impact method is used, due to the characteristics of the gas product, it cannot fully contact the culture medium, making it difficult to accurately reflect the number of living microorganisms in the gas product. In addition, according to the sampling time and sampling volume requirements, the long-term impact of the gas will cause the surface of the culture medium to dry out, resulting in poor adhesion ability, and the microorganisms are easily dried out and killed. Accurate counting results cannot be obtained after cultivation.

[0005] Existing gas collectors, such as straight-line impact gas collectors, have a small contact surface and high impact force, which easily generates aerosols. This causes microorganisms to adhere to the collector walls along with the collected liquid, resulting in low retention rates and inaccurate test results. Flue gas collectors, while suitable for flue gas sampling, are unsuitable for microbial sampling because they require filtering particulate impurities from the gas through a sand core, which microorganisms cannot pass through.

[0006] The existing microbial limit testing methods in the 2020 edition of the Chinese Pharmacopoeia include: the plate method: directly taking a certain volume of the drug and mixing it with the culture medium, then incubating it and counting the number of colonies formed. This method is not applicable to gaseous drugs; the membrane filtration method: removing the antibacterial components in the sample by filtration, and then placing the filter membrane on the culture medium for incubation and counting. However, it is only applicable to liquid samples; the most probable number method (MPN method): only applicable to samples with very small amounts of microbial contamination, estimating the number of microorganisms through serial dilution and incubation. It is not applicable to APIs for injections, which have strict quality standards for preparations. None of the above pharmacopoeial methods are applicable to the testing of drugs with gaseous properties. The existing technology also does not disclose an effective and compatible collection system and collection method for microorganisms in pharmaceutical gases. Summary of the Invention

[0007] The purpose of the present invention is to provide a collection system and method for microorganisms in medicinal gas, which are convenient and fast, easy to sterilize, and have high microorganism survival rate, high interception rate and high accuracy.

[0008] To achieve the first objective of the present invention, the technical solution is: a system for collecting microorganisms in pharmaceutical gases, comprising a flow controller 21 and a collector 31. The bottom of the flow controller 21 is connected to a gas cylinder adapter 12 via a pressure reducing valve 11. A gas pipe at the top of the flow controller 21 is connected to the collector 31. The collector 31 is equipped with a stopper 34, through which a gas pipe 32 extends to the bottom of the collector 31. The bottom of the collector 31 is filled with a collection liquid 33. The tail of the gas pipe 32 is designed to be spiral, and the outer side of the spiral pipe 36 is provided with a plurality of air holes 37. The top of the collector 31 is provided with an outlet pipe 35 connected to a check valve 38. The collection liquid is composed of a mixture of medical glycerin and 0.9% sterile sodium chloride solution in a volume ratio of 1:1. The collector is primarily used for capturing and collecting microorganisms, reducing aerosol generation and reducing liquid splashing and adhesion.

[0009] Furthermore, the flow controller and the pressure reducing valve are connected by a hard pipe, preferably a 316L BA grade stainless steel pipe, which has strong stability, is not easy for microorganisms to attach and grow, and can be sterilized by high temperature steam.

[0010] Furthermore, the flow controller and the collector are connected by a soft tube, preferably a silicone hose, which can be sterilized by high-temperature steam and has no biological toxicity.

[0011] Furthermore, the flow controller is preferably a corrosion-resistant, flow-adjustable glass rotor flowmeter.

[0012] Furthermore, the valve body of the pressure reducing valve is preferably made of clean material 316L stainless steel, and the valve stem filler is preferably PTFE. The purpose of setting up the pressure reducing valve is to reduce pressure, reduce damage, and retain microbial activity.

[0013] Furthermore, the collector is preferably made of high borosilicate glass, which is resistant to high temperatures, highly transparent, acid-resistant and corrosion-resistant.

[0014] Furthermore, the check valve is preferably made of PTFE to prevent tail contamination.

[0015] Furthermore, the collector has a thickened ground edge with fine ground edges, tight docking and good sealing performance; the collector bottle stopper is preferably made of silicone stopper with good sealing and high temperature resistance.

[0016] Furthermore, the collector preferably adopts a round bottle with a base diameter larger than the bottle body diameter, which has good stability and is not easy to tip over.

[0017] To achieve the above second object of the invention, the present invention provides a method for collecting microorganisms in medicinal gas, comprising the following steps: (1) Prepare the collection liquid, add the collection liquid into the collector, and place the collector containing the collection liquid into a high-temperature steam sterilizer for sterilization; (2) Disinfect the environment around the medicinal gas cylinder, impurities at the bottle mouth, and the inlet and outlet of the pressure reducing valve; (3) Connect the medicinal gas bottle to the collection system, displace the gas in the pipeline, and make the gas flow out from the flow meter outlet; (4) Connecting the collector: Disinfect the flow controller outlet and its surroundings, and connect the flow controller to the collector with sterilized pipes; (5) Adjust the pressure reducing valve and flow meter so that the medicinal gas can be trapped by the collecting liquid; (6) The collected liquid is injected into agar culture medium for cultivation to determine the number of microorganisms in the medicinal gas.

[0018] The bottom end of the collector of the present invention is a spiral structure, which can increase the contact between the gas and the collected liquid and improve the interception efficiency. The air holes on the outside of the spiral reduce the impact force on the liquid, making it difficult for aerosols to adhere to the collector wall. A check valve is installed at the tail end of the collector to avoid external contamination. The present invention uses a mixture of medical glycerin and 0.9% sterile sodium chloride solution in a specific ratio as the collection liquid to intercept microorganisms, with a high recovery rate. This solves the problem that glycerin is used to preserve microorganisms and easily causes the activity of microorganisms to decrease. At the same time, it solves the problem that 0.9% saline has poor interception effect and is prone to splashing.

[0019] The system and method for collecting microorganisms in medicinal gas provided by the present invention have a high collection retention rate, a gentle collection process, can maximize the retention of microbial activity in the sample, are highly accurate, and are simple to operate. They solve the problems of collecting and counting microorganisms in medicinal gas and have high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is an overall schematic diagram of the collection system of the present invention.

[0021] Figure 2 It is an overall schematic diagram of the collector in the present invention.

[0022] Figure 3 This is a diagram of the internal structure of the collector in the present invention.

[0023] Among them: 11. Pressure reducing valve, 12. Gas cylinder adapter, 21. Flow controller, 31. Collector, 32. Air pipe, 33. Collecting liquid, 34. Bottle stopper, 35. Air outlet pipe, 36. Spiral tube, 37. Air hole, 38. Check valve. DETAILED DESCRIPTION

[0024] The collection scheme of the present invention will be clearly and completely described below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] like Figures 1 to 3 As shown, a system for collecting microorganisms in medicinal gas includes a flow controller 21 and a collector 31. The bottom of the flow controller 21 is connected to the gas cylinder adapter 12 through a pressure reducing valve 11. The air pipe on the upper part of the flow controller 21 is connected to the collector 31. The collector 31 is equipped with a bottle stopper 34. The air pipe 32 extends into the bottom of the collector 31 through the bottle stopper 34. The bottom is filled with a collecting liquid 33. The tail part of the air pipe 32 is designed to be spiral. A plurality of air holes 37 are opened on the outer side of the spiral tube 36. An air outlet pipe 35 is opened on the upper part of the collector 31 and is connected to a check valve 38. The collecting liquid 33 is composed of a mixture of medical glycerin and 0.9% sterile sodium chloride solution in a volume ratio of 1:1.

[0027] The flow controller 21 and the pressure reducing valve 11 are connected by a 316L BA grade stainless steel pipe, which has strong stability, makes it difficult for microorganisms to attach and grow, and can be sterilized by high-temperature steam; the flow controller 21 and the collector 31 are connected by a silicone hose, which can be sterilized by high-temperature steam and has no biological toxicity; the flow controller 21 uses a glass rotor flowmeter; the valve body of the pressure reducing valve 11 is made of clean material 316L, and the valve stem filling material is PTFE; the collector 31 is made of high borosilicate glass, which is resistant to high temperature, high transparency, acid and corrosion; the check valve 38 is made of PTFE to prevent tail contamination; the collector 31 has a thickened ground mouth with fine ground mouth, tight docking and good sealing performance; the collector bottle stopper 34 is a silicone stopper with good sealing and high temperature resistance; the collector 31 uses a round bottle with a base diameter larger than the bottle body diameter, which has good stability and is not easy to tip over.

[0028] Example 2

[0029] A method for collecting microorganisms in medicinal gas comprises the following steps: (1) Adding collection liquid: Open the silicone plug on the collector and inject the prepared collection liquid at an angle; (2) Place the collector containing the collected liquid in step (1) in a high-temperature steam sterilizer at 121°C for 30 minutes for high-temperature wet sterilization to ensure that the collector is sterile; (3) Use disinfectant to spray the environment around the gas cylinder to disinfect it to ensure that the operation process does not introduce environmental microbial contamination; (4) Cleaning the bottle mouth: Remove the dust cap of the gas cylinder, gently open and close the bottle valve to replace it, blow away the impurities at the bottle mouth, and visually check for dust and dirt; (5) Disinfection of the connection port: Use a cotton swab dipped in 75% alcohol to wipe the valve port of the gas cylinder and the inlet and outlet of the pressure reducing valve for disinfection. After disinfection, wipe them dry to prevent alcohol residue; (6) Connect the pressure reducing valve: one end of the pressure reducing valve is connected to the gas cylinder, and the other end is connected to the inlet of the glass rotor flowmeter; the middle is connected with a 316L stainless steel pipe; (7) Replacement pipeline: slightly open the cylinder valve, adjust the pressure reducing valve, let the gas flow out from the flow meter outlet, blow for several minutes, and replace the pipeline; (8) Connecting the collector: Spray disinfectant on the outlet of the glass rotor flowmeter and its surroundings, connect the sterilized silicone hose, and connect the other end of the hose to the collector inlet; (9) Slowly adjust the bottle valve, pressure reducing valve, and flow meter to control the flow so that the gas slowly passes through the collecting liquid and is trapped; (10) Take the collected liquid on the operating table in the clean area, inject it into the agar culture medium, place it at a suitable temperature for cultivation, and determine the number of microorganisms in the medicinal gas.

[0030] Example 3 The experimental reagents and equipment are shown in Tables 1 and 2.

[0031]

[0032]

[0033] Experimental strain and number: Escherichia coli [ATCC25922] The above-mentioned strains are provided by the China Food and Drug Inspection Institute, and the number of passages should not exceed 5 generations.

[0034] Experimental process: Preparation of standard bacterial solution: A nutrient agar slant culture of Escherichia coli cultured at 36±1°C for 18-24 hours was diluted 10-fold with sterile sodium chloride-peptone buffer, pH 7.0, to prepare a bacterial suspension of appropriate concentration for use. The bacterial suspension with a colony count of 10-100 cfu / ml after culturing at 36±1°C for 5 days was selected, as shown in Table 3.

[0035] If the standard bacterial solution is kept at room temperature after preparation, it should be used within 2 hours; if it is stored at 2-8°C, it can be used within 24 hours.

[0036]

[0037] The collection solution was prepared according to the proportions in Table 4 (120 ml per collection solution).

[0038]

[0039] Preparation for sterilization of the collection fluid: Take 10 250ml screw-cap bottles and prepare 2 parallel copies for each group according to the ratio in Table 4 (one copy is used as a positive control and the other copy is used as a negative control). Also prepare 5 collectors (test group) according to the ratio in Table 4 and perform wet sterilization.

[0040] Other auxiliary supplies are sterilized.

[0041] Petri dish pouring method Test Groups: Samples were collected from five test groups using the collection system of Example 1 (operated in the positive inoculation chamber). 1 ml of standard bacterial solution was added to the aerosol generator of each group. 1.0 ml of the bacterial collection solution was injected into a sterile plate (two plates were prepared in parallel for each group), followed by 15-20 ml of MacConkey agar culture medium maintained at a temperature not exceeding 60°C.

[0042] Positive control group: Add 1 ml of standard bacterial solution to each of five screw-cap bottles (prepared with the collection solution in the corresponding ratio). Transfer 1.0 ml of the collection solution containing bacteria to a sterile plate (prepare two parallel plates for each group) and add 15-20 ml of MacConkey agar medium not exceeding 60°C.

[0043] Negative control group: 5 screw-cap bottles (prepared with collection solution in the corresponding ratio). Take 1.0 ml of the collection solution from each group and inject it into a sterile plate (prepare 2 plates in parallel for each group), and then inject 15-20 ml of MacConkey agar medium at a temperature not exceeding 60°C.

[0044] The above-mentioned plates were cultured at 36±1°C for no more than 5 days.

[0045] Repeat the above steps three times.

[0046] The collection rate results are shown in Table 5, Table 6, and Table 7. Note: (Collection rate = number of colonies in the test group / number of colonies in the positive control group)

[0047]

[0048]

[0049] As can be seen from the table above, the bacterial collection rates of test groups 2 / 3 / 4 / 5 are in the range of 0.9-1.0. Since glycerol has a strong dehydrating property, it will cause the microorganisms to dehydrate and die. Therefore, the ratio of the collection liquid is medical glycerol: 0.9% sterile sodium chloride = 1:1.

[0050] Summary: The parameters involved in the collection system and collection plan are: collection fluid: 120ml; collection fluid ratio: medical glycerin: 0.9% sterile sodium chloride = 1:1 Example 4 Test items and acceptance criteria are shown in Table 8.

[0051]

[0052] A method for collecting microorganisms in medicinal gas comprises the following steps: (1) Adding the collection solution: Open the silicone stopper on the collector and inject 120 ml of the collection solution prepared according to the method at an angle; (2) Place the collector containing the collected liquid in a high-temperature steam sterilizer at 121°C for 30 minutes for high-temperature wet sterilization to ensure that the collector is sterile; (3) Use disinfectant to spray the environment around the gas cylinder for secondary disinfection; (4) Cleaning the bottle mouth: Remove the dust cap of the gas cylinder, gently open and close the bottle valve 10 times, blow out impurities from the bottle mouth, and visually check for dust and dirt; (5) Disinfection of the connection port: Use a cotton swab dipped in 75% alcohol to wipe the valve port of the gas cylinder and the inlet and outlet of the pressure reducing valve for disinfection. After disinfection, wipe them dry to prevent alcohol residue; (6) Connect the pressure reducing valve: one end of the pressure reducing valve is connected to the gas cylinder, and the other end is connected to the inlet of the glass rotor flowmeter; the middle is connected with a 316L stainless steel pipe; (7) Replacement pipeline: slightly open the valve of the gas cylinder, adjust the pressure reducing valve to make the gas flow out from the outlet of the flow meter, blow for 10 minutes, and replace the pipeline; (8) Connecting the collector: Spray disinfectant on the outlet of the glass rotor flowmeter and its surroundings, connect the sterilized silicone hose, and connect the other end of the hose to the collector inlet; (9) Slowly adjust the bottle valve, pressure reducing valve, and flow meter to control the flow rate to 100 ml / min, allowing the gas to slowly pass through the collection liquid and be trapped for 40 minutes; this serves as the test solution; (10) Open the biosafety cabinet; (11) Test group (total aerobic bacterial count): Use the plate pouring method to take 10 ml of the test solution and place it in a sterile small test tube. Then add no more than 100 μl of Escherichia coli and mix well so that the bacterial count per 1 ml of the test solution is no more than 100 cfu. Take 1.0 ml of the test solution containing bacteria and inject it into a sterile plate (prepare 2 plates in parallel for the test group), and then inject 15-20 ml of MacConkey agar medium with a temperature not exceeding 60°C; (11) Bacterial solution control group: replace the test solution with the collected solution and perform the same operation as the experimental group, preparing two plates in parallel; (12) Negative control group: replace the test solution with the collected solution and perform the same operation as the test group, but do not add bacterial solution, and prepare two parallel plates; (13) Test sample control group: Take 10 ml of the test solution and replace the bacterial solution with the collection solution. Perform the same operation as the test group and prepare two parallel plates.

[0053] The above-mentioned plates were cultured at 36±1°C for no more than 5 days.

[0054] Repeat the above process 3 times.

[0055] The recovery rate determination results and judgment are shown in Table 9.

[0056]

[0057] As can be seen from Table 9, the bacterial count recovery rate of the experimental group is within the range of 0.5-2.0 specified in the 2020 edition of the Chinese Pharmacopoeia. Therefore, the collector, collection system and collection scheme of microorganisms in medicinal gas can be carried out using this method.

[0058] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A system for collecting microorganisms in medicinal gas, characterized in that: The invention comprises a flow controller (21) and a collector (31). The bottom of the flow controller (21) is connected to a gas cylinder adapter (12) via a pressure reducing valve (11). The upper air pipe of the flow controller (21) is connected to the collector (31). The collector (31) is provided with a bottle stopper (34). The air pipe (32) penetrates into the bottom of the collector (31) through the bottle stopper (34). The bottom is filled with a collecting liquid (33). The tail of the air pipe (32) is designed to be spiral. The outer side of the spiral pipe (36) is provided with a plurality of air holes (37). The upper part of the collector (31) is provided with an air outlet pipe (35) and is connected to a check valve (38). The collecting liquid is composed of a mixture of medical glycerin and 0.9% sterile sodium chloride solution in a volume ratio of 1:

1.

2. The collection system according to claim 1, characterized in that The flow controller and the pressure reducing valve are connected by 316L BA grade stainless steel pipe.

3. The collection system according to claim 1, characterized in that The flow controller and the collector are connected by a silicone hose.

4. The collection system according to claim 1, characterized in that The flow controller uses a glass rotor flowmeter.

5. The collection system according to claim 1, characterized in that The valve body of the pressure reducing valve is made of 316L stainless steel and the valve stem filling material is PTFE.

6. The collection system according to claim 1, characterized in that The collector is made of high borosilicate glass.

7. The collection system according to claim 1, characterized in that The material of the check valve is PTFE.

8. The collection system according to claim 1, characterized in that The bottle stopper is a silicone stopper.

9. The collection system according to claim 1, characterized in that The collector adopts a round bottle with a base diameter larger than the bottle body diameter.

10. A collection method of a collection system according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Prepare the collection liquid, add the collection liquid into the collector, and place the collector containing the collection liquid into a high-temperature steam sterilizer for sterilization; (2) Disinfect the environment around the medicinal gas cylinder, impurities at the bottle mouth, and the inlet and outlet of the pressure reducing valve; (3) Connect the medicinal gas bottle to the collection system, displace the gas in the pipeline, and make the gas flow out from the flow meter outlet; (4) Connecting the collector: Disinfect the outlet of the flow controller and its surroundings, and connect the flow controller to the collector with sterilized pipes; (5) Adjust the pressure reducing valve and flow meter so that the medicinal gas can be trapped by the collecting liquid; (6) The collected liquid is injected into agar culture medium for cultivation to determine the number of microorganisms in the medicinal gas.

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