Sheet type medicine packing material microorganism counting and sampling method

Abstract

The invention provides a microbiological counting and sampling method for a sheet medicine packing material, which comprises the following steps: firstly, preparing a casein tryptone agar culture medium (TSA) contact dish and a Saborne glucose agar culture medium (SDA) contact dish, then carrying out pressing sampling, and carrying out non-sterile product microbiological limit inspection according to general rules 1105 of four parts of Chinese Pharmacopoeia in 2015 edition: carrying out transfer culture by a microbiological counting method; and observing and counting. According to the contact disc sampling method, the surface of the sheet medicine packing material is in close contact with the culture medium, so that the pollution microorganisms are fully adhered to the culture medium, the collection rate and the detection rate are improved, the recovery rate is greater than 80%, a test solution does not need to be prepared, the method belongs to a direct sampling method, the operation is simple, the sampling area is accurate, one-step sampling is realized, and the secondary pollution probability is reduced; the defects of an existing wiping method are avoided, inspection data are more accurate and reliable, the microbial contamination condition of the sheet type medicine packing material can be truly reflected, meanwhile, time and cost are saved, and the experiment efficiency is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug microbial testing, and in particular relates to a method for sampling and counting microorganisms of sheet-type drug packaging materials. Background Art

[0002] Pharmaceutical packaging materials refer to the packaging materials and containers used by pharmaceutical manufacturers and medical institutions for direct contact with drugs. They can be classified into plastic, metal, glass, ceramic, rubber and other types (such as paper, desiccant) by material, and can also be composed of two or more materials (such as composite film, aluminum-plastic composite cover, etc.); according to use and shape, they can be divided into infusion bottles (bags, films and accessories), ampoules, medicinal (injection, oral or external dosage form) bottles (tubes, covers), medicinal rubber stoppers, medicinal prefilled syringes, medicinal eye (nose, ear) drop bottles, medicinal hard sheets (films), medicinal aluminum foils, medicinal ointment tubes (boxes), medicinal spray (aerosol) pumps (valves, cans), medicinal desiccant, etc. In recent years, with the improvement of the state's supervision of pharmaceutical packaging materials, higher requirements have been placed on the quality of pharmaceutical packaging materials. However, when conducting microbial inspections on pharmaceutical packaging materials, due to the different properties of various materials and containers, there are differences in sampling rules, sampling quantity, test solution preparation methods, inspection methods, judgment criteria, etc., which affects the reliability of the results.

[0003] When sampling for microbial counting, the existing sheet-type pharmaceutical packaging materials generally adopt the wiping method in accordance with the provisions of the national pharmaceutical packaging material standards YBB00132002-2015 "General Rules for Pharmaceutical Composite Films and Bags" and YBB00152002-2015 "Pharmaceutical Aluminum Foil". However, it is found in actual inspection work that the existing wiping sampling method has many disadvantages. For example, the wiping method for testing the microbial counting of sheet materials requires the preparation of the test solution first, and further membrane filtration to detect the number of microorganisms, which belongs to an indirect sampling method. At the same time, repeated wiping with the same cotton swab may have the risk of cross-contamination, and the sampling area is not accurate, resulting in large errors in the experimental results and low detection efficiency. In addition, the number of microorganisms collected by the wiping method is affected by multiple subjective and objective factors such as the wiping method, wiping strength, tools used, and sampled objects of the experimenter, which will result in poor reproducibility and low accuracy of the test results, resulting in the experimental results may not be consistent with the actual pollution situation. These are all factors that reduce the accuracy of the test results. At the same time, relevant literature also reports that the removal rate and recovery rate of microorganisms by the wiping method are low.

[0004] The contact dish or contact plate sampling method is usually used to monitor environmental surface microorganisms, such as the detection of surface microorganisms in clean rooms, laboratories or hospitals, but these are mostly on smooth and hard carrier surfaces such as stainless steel and glass, and there are no reports on its application in the detection of microorganisms in pharmaceutical packaging materials. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a microbial counting and sampling method for sheet-type pharmaceutical packaging materials in view of the above-mentioned deficiencies in the prior art, thereby avoiding the influence of the existing sampling method on the experimental results, improving the collection rate and detection rate, and being able to truly reflect the bacterial status of the surface of the object.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for sampling and counting microorganisms of sheet-type pharmaceutical packaging materials, the method comprising the following steps:

[0008] S1. Prepare the culture medium contact plate

[0009] Prepare 4 contact plates of tryptic soy peptone agar medium and 4 contact plates of Sabouraud dextrose agar medium. The height of the culture medium protruding from the plate mouth is 2 mm. The sampling area of ​​each contact plate is 25 cm. 2 ;

[0010] S2. Sampling

[0011] S201, aerobic bacteria count sampling: Take 4 tryptic soy agar contact plates obtained in S1, remove the plate cover, and sample at any 4 points on the surface of the sheet drug packaging material, with a total sampling area of ​​100cm 2 , press the convex surface of the culture medium against the surface of the drug packaging material for no less than 10 seconds, apply constant and uniform pressure during pressing, and no circular or linear movement is allowed. The pressing force should be appropriate so that the culture medium does not deform, ensuring that the convex surface of the culture medium is in close contact with the surface of the drug packaging material;

[0012] S202, sampling for mold and yeast count: use the four Sabouraud dextrose agar medium contact plates obtained in S1 to sample the sheet-type pharmaceutical packaging materials, using the same method as S201;

[0013] S3. Transfer, culture and counting

[0014] Cover the sampled tryptic soy peptone agar contact plate and Sabouraud dextrose agar contact plate, and transfer culture according to 1105 Microbial Limit Test for Non-sterile Products: Microbial Count Method of Part IV of the 2015 edition of the Chinese Pharmacopoeia, and observe the count.

[0015] Preferably, the sheet-type drug packaging material includes a pharmaceutical film, a pharmaceutical hard sheet and a pharmaceutical aluminum foil.

[0016] Preferably, the tryptic soytone agar medium contact plate and the Sabouraud dextrose agar medium contact plate in S1 are obtained by pouring the tryptic soytone agar medium and the Sabouraud dextrose agar medium into disposable culture dishes, respectively.

[0017] Preferably, the tryptic soy peptone agar medium contact plate in S4 is used for aerobic bacteria culture, the culture temperature is 30-35°C, and the culture time is 5 days; the Sabouraud dextrose agar medium contact plate is used for mold and yeast culture, the culture temperature is 20-25°C, and the culture time is 7 days.

[0018] The present invention has the following significant technical effects:

[0019] The present invention provides a method for sampling microorganisms for sheet-type pharmaceutical packaging materials, which avoids the drawbacks of the existing national standard wiping method, makes the test data more accurate and reliable, can truly reflect the microbial contamination status of sheet-type pharmaceutical packaging materials, and has a recovery rate of more than 80%. Compared with the wiping method, the method of the present invention has the following advantages for sampling microorganisms for pharmaceutical packaging materials:

[0020] (1) The microbial counting sampling of the sheet-type drug packaging material of the present invention achieves close contact between the surface of the sheet-type object and the culture medium, ensuring that the contaminated microorganisms are fully adhered to the culture medium, improving the collection rate and detection rate, and the experimental results can truly reflect the bacterial status of the surface of the object;

[0021] (2) The wiping method for testing the microbial count of sheet-type pharmaceutical packaging materials requires the preparation of a test solution first, and then the membrane filtration is used to detect the number of microorganisms, which is an indirect sampling method; the sampling method of the present invention is used for sampling microorganisms of sheet materials for pharmaceutical packaging, which does not require the preparation of a test solution, and is a direct sampling method, thereby saving experimental time and consumables costs, and improving experimental efficiency;

[0022] (3) The sampling method of the present invention is simple to operate, does not require the aid of a sterile mold, has an accurate sampling area, and only requires one step for sampling, thereby optimizing the experimental steps, reducing influencing factors, reducing the probability of secondary contamination, and ensuring the reliability of the experimental results.

[0023] (4) The sampling method of the present invention is highly operable, has a simple process, does not require high technical skills from the experimenter, and is suitable for use by laboratory personnel at all levels.

[0024] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a photo of a tryptic soy peptone agar medium (TSA) contact plate and a Sabouraud dextrose agar medium (SDA) contact plate of Example 1, wherein Figure a is a tryptic soy peptone agar medium (TSA) contact plate, and Figure b is a Sabouraud dextrose agar medium (SDA) contact plate;

[0026] Figure 2 is a sampling diagram of Example 1;

[0027] Figure 3 This is a wiping method sampling diagram of Comparative Example 1;

[0028] Figure 4 This is the sampling diagram of the elution method of Comparative Example 2;

[0029] Figure 5 This is a direct inoculation sampling diagram of Comparative Example 3;

[0030] Figure 6 Schematic diagram of membrane filtration in Comparative Examples 1 to 3;

[0031] Figure 7 The recovery results of the four sampling methods on the pharmaceutical film of Example 2;

[0032] Figure 8 The recovery results of the four sampling methods on the medicinal hard tablets of Example 2;

[0033] Fig. 9 These are the recovery results of the four sampling methods on the medicinal aluminum foil in Example 2. DETAILED DESCRIPTION

[0034] Example 1

[0035] This embodiment is a method for sampling and counting microorganisms in sheet-type pharmaceutical packaging materials, which includes the following steps:

[0036] S1. Prepare the culture medium contact plate

[0037] Prepare 4 TSA contact plates and 4 SDA contact plates, with the culture medium protruding 2 mm from the plate mouth, an inner diameter of 56 mm, and an area of ​​25 cm. 2 ; TSA was used for aerobic bacterial culture, and SDA was used for fungus and yeast culture.

[0038] The tryptic soy peptone agar medium contact plate and Sabouraud dextrose agar medium contact plate used were homemade in the laboratory, and were obtained by pouring the tryptic soy peptone agar medium and Sabouraud dextrose agar medium into disposable culture dishes, respectively. Among them, the disposable culture dish (inner diameter 56 mm) was purchased from Qingdao Jindian Biochemical Equipment Co., Ltd.; the tryptic soy peptone agar medium and Sabouraud dextrose agar medium were purchased from Beijing Sanyao Technology Co., Ltd. and prepared according to the instructions.

[0039] S2. Sampling

[0040] S201. Take 4 TSA contact plates and sample 4 points on the surface of the sheet drug packaging material that are easily contaminated. The total sampling area is 100 cm. 2The method is: remove the dish cover, press the raised surface of the culture medium on the surface of the drug packaging material for not less than 10 seconds, apply constant and uniform pressure when pressing, and no circular or linear movement is allowed. The pressure should be strong enough to prevent the culture medium from deforming, ensuring that the raised surface of the culture medium is in close contact with the surface of the drug packaging material.

[0041] S202. Sample the sheet-type pharmaceutical packaging material using Sabouraud dextrose agar (SDA) contact plate, using the same method as S201.

[0042] S3. Transfer, culture and counting

[0043] Cover the sampled tryptic soy agar (TSA) contact plates and Sabouraud dextrose agar (SDA) contact plates, and perform transfer culture according to the 2015 edition of the Chinese Pharmacopoeia, Part IV, General Rules 1105, Microbial Limit Test for Non-sterile Products: Microbial Count Method, and observe the count.

[0044] Figure 1 The figures are the contact plates of tryptic soy agar (TSA) medium (a) and Sabouraud dextrose agar (SDA) medium (b) of this example.

[0045] Figure 2 2 is a contact plate sampling diagram of the present embodiment.

[0046] Comparative Example 1

[0047] The wiping method is used to collect samples and count the microorganisms of sheet-type pharmaceutical packaging materials, which specifically includes the following steps:

[0048] Prepare one TSA and one SDA sterile plate (90 mm in diameter). Take a sterile cotton swab and slightly wet it with sodium chloride injection. Use one cotton swab to wipe the surface of the drug packaging material 5 times, then use another cotton swab to wipe it 5 times. Use 2 cotton swabs to wipe each position 10 times in total, and wipe 5 positions with a total area of ​​100 cm. 2 Cut each cotton swab immediately after wiping, and put the head of the cotton swab into a conical bottle containing 30mL of sodium chloride injection. After all the cotton swabs are put into the bottle, shake the conical bottle quickly for 1 minute and let it stand for 10 minutes to obtain the test solution. After the test solution is filtered through a membrane, stick the filter membrane with the bacterial surface facing up on the TSA or SDA plate culture medium (TSA and SDA are sampled 100cm each). 2 ), according to the 2015 edition of the "Chinese Pharmacopoeia" Part IV General Chapter 1105 Microbial Limit Test for Non-sterile Products: Microbial Count Method for cultivation and observation of counts.

[0049] Figure 3 This is a sampling diagram using the wipe method.

[0050] Comparative Example 2

[0051] The leaching method is used to count the microorganisms of sheet-type pharmaceutical packaging materials, which specifically includes the following steps:

[0052] Prepare one TSA and one SDA sterile plate (90 mm in diameter). Pick up the drug packaging material with sterile tweezers, place a sterile container at the bottom, draw 30 mL of sodium chloride injection with a sterile syringe, rinse the entire contaminated surface of the drug packaging material at a uniform speed, collect all the eluent, and the test solution is obtained. After the test solution is filtered through a membrane, stick the filter membrane with the bacterial surface facing up on the TSA or SDA plate culture medium (100 cm for each TSA and SDA sample). 2 ), according to the 2015 edition of the "Chinese Pharmacopoeia" Part IV General Chapter 1105 Microbial Limit Test for Non-sterile Products: Microbial Count Method for cultivation and observation of counts.

[0053] Figure 4 This is the sampling diagram of the elution method.

[0054] Comparative Example 3

[0055] The direct inoculation method is used to count the microorganisms of sheet-type pharmaceutical packaging materials, which specifically includes the following steps:

[0056] Prepare one TSA and one SDA sterile plate (90 mm in diameter). Use sterile scissors to cut an area of ​​100 cm 2 Cut the sheet medicine packaging material into pieces of about 3 to 4 cm 2 Put all the small pieces of the sample into a conical bottle containing 30mL of sodium chloride injection. After cutting, shake the bottle quickly for 1 minute and absorb all the solution to obtain the test solution. After the test solution is filtered through a membrane, stick the filter membrane with the bacterial surface facing up on the TSA or SDA plate culture medium (100cm for each TSA and SDA sample). 2 ), according to the 2015 edition of the "Chinese Pharmacopoeia" Part IV General Chapter 1105 Microbial Limit Test for Non-sterile Products: Microbial Count Method for cultivation and observation of counts.

[0057] Figure 5 This is a sampling diagram for direct inoculation method.

[0058] Figure 6 It is a schematic diagram of the membrane filtration process of Comparative Examples 1 to 3.

[0059] The microorganism counts of the sheet-type drug packaging material samples were performed using different methods of Example 1 and Comparative Examples 1 to 3, and the total number of aerobic bacteria, molds and yeasts obtained from the two culture media of TSA and SDA using the four sampling methods were calculated. The results are shown in Tables 1 and 2:

[0060] Table 1 Results of aerobic microbial counts (100 cm 2 )

[0061]

[0062]

[0063] Table 2 Results of mold and yeast microbial counts collected by different sampling methods (100 cm 2 )

[0064]

[0065]

[0066] Example 2

[0067] In this example, a sample of a sheet-type drug packaging material with artificial spike contamination was used for microbial counting sampling and recovery calculation. The specific method is as follows:

[0068] The strains used included Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger, all of which were obtained from Beijing Sanyao Technology Co., Ltd. Quantitative strains with a content of 0.5-1.0×10 3 cfu / g, add the bacterial solution into the strain bottle and vortex mix for 30s to obtain the experimental bacterial solution.

[0069] Preparation area is 100cm 2 5 square (10cm×10cm) medical films were sterilized before the experiment. The samples were placed on sterilized kraft paper on the operating table of the biosafety cabinet. 100μL (i.e. 50-100cfu) of Staphylococcus aureus suspension was applied to the upward side of the medical films. A disposable inoculation loop was used to inoculate the suspension on 100cm 2 The first artificially contaminated sample was discarded, and the remaining four artificially contaminated samples were sampled for microorganisms on their surfaces using the method described in Example 1, and the wiping method, rinsing method, and direct inoculation method described in Comparative Examples 1 to 3, respectively, to obtain the number of colonies counted by different sampling methods and calculate the recovery rate.

[0070] Bacteria liquid group test: Take 100μL of Staphylococcus aureus suspension and inoculate it into a conical bottle of 30mL sodium chloride injection, shake it quickly for 1 minute, and let it stand for 10 minutes to obtain the test solution. After the test solution is filtered by membrane, check the colony count of the bacteria liquid group according to the general rules 1105 of the fourth part of the 2015 edition of the Chinese Pharmacopoeia.

[0071] Calculate the recovery rate for different sampling methods:

[0072]

[0073] Artificial contamination samples of Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger were prepared in the same way (two samples were prepared for Candida albicans and Aspergillus niger, one for SDA detection, and the bacterial liquid group was detected by SDA simultaneously). Each strain was detected by four sampling methods, and the corresponding recovery rate was calculated. Each test strain was tested independently for 6 times in parallel, and the average of the 6 test results was used.

[0074] Artificially spiked and contaminated pharmaceutical hard tablets and pharmaceutical aluminum foil samples were prepared in the same manner as above for microbial count sampling.

[0075] The recovery results of the four sampling methods on pharmaceutical film, pharmaceutical hard tablet and pharmaceutical aluminum foil are as follows: Figure 7 , Figure 8 and Fig. 9 As shown, the results show that: the method of the present invention (contact plate sampling) > direct inoculation method > wiping method > rinsing method, and the recovery rate of the method of the present invention is greater than 80%, which is the highest recovery rate, indicating that this method can obtain a higher microbial capture rate when used for packaging sheet sampling. There is a significant difference in the recovery rate between the method of the present invention and the wiping method and the rinsing method P<0.05; the recovery rate of the method of the present invention is slightly higher than that of direct inoculation, but there is no significant difference between the two P>0.05. There is no significant difference in the recovery rate of the four sampling methods in pharmaceutical film, pharmaceutical aluminum foil and pharmaceutical hard sheet P>0.05, which shows that there is no difference in the collection rate of the three sheets among the sampling methods.

[0076] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for sampling and counting microorganisms of sheet-type pharmaceutical packaging materials, characterized in that: The method comprises the following steps: S1. Prepare the culture medium contact plate Prepare 4 contact plates of tryptic soy peptone agar medium and 4 contact plates of Sabouraud dextrose agar medium. The height of the culture medium protruding from the plate mouth is 2 mm. The sampling area of ​​each contact plate is 25 cm. 2 ; S2. Sampling S201, aerobic bacteria count sampling: Take 4 tryptic soy agar contact plates obtained in S1, remove the plate cover, and sample at any 4 points on the surface of the sheet drug packaging material, with a total sampling area of ​​100cm 2 , press the convex surface of the culture medium against the surface of the drug packaging material for no less than 10 seconds, apply constant and uniform pressure during pressing, and no circular or linear movement is allowed. The pressing force should be appropriate so that the culture medium does not deform, ensuring that the convex surface of the culture medium is in close contact with the surface of the drug packaging material; S202, sampling for mold and yeast count: use the four Sabouraud dextrose agar medium contact plates obtained in S1 to sample the sheet-type pharmaceutical packaging materials, using the same method as S201; S3. Transfer, culture and counting Cover the sampled tryptic soy peptone agar contact plate and Sabouraud dextrose agar contact plate, and transfer culture according to 1105 Microbial Limit Test for Non-sterile Products: Microbial Count Method of Part IV of the 2015 edition of the Chinese Pharmacopoeia, and observe the count.

2. A method for sampling and counting microorganisms of sheet-type pharmaceutical packaging materials according to claim 1, characterized in that: The sheet-type medicine packaging materials include medicinal films, medicinal hard sheets and medicinal aluminum foils.

3. A method for sampling and counting microorganisms of sheet-type pharmaceutical packaging materials according to claim 1, characterized in that: The tryptic soytone agar medium contact plate and the Sabouraud dextrose agar medium contact plate in S1 are obtained by pouring the tryptic soytone agar medium and the Sabouraud dextrose agar medium into disposable culture dishes, respectively.

4. A method for sampling and counting microorganisms of sheet-type pharmaceutical packaging materials according to claim 1 or 3, characterized in that: S4 The tryptic soy peptone agar medium contact plate is used for aerobic bacteria culture, the culture temperature is 30-35°C, and the culture time is 5 days; the Sabouraud dextrose agar medium contact plate is used for mold and yeast culture, the culture temperature is 20-25°C, and the culture time is 7 days.

Images