The experimental principle, preparation and operation steps of the aseptic examination method of biological products?

1. Purpose: to establish a standard operating procedure for sterility checking to ensure the accuracy of the test results. 2. 2. Scope: applicable to the quality control section of the plant's laboratory on the plant production of injections for sterility check. 3. Responsibility: the laboratory technician has the responsibility to operate in accordance with these operating procedures, and is responsible for the results of the test. 4. Definition: aseptic inspection method refers to a method of checking whether the drug is sterile. 5. 5.1 Aseptic operation equipment: aseptic operation room or ultra-clean workbench, aseptic gowns, masks, hats, disinfectant shoes. 5.1.1 Aseptic operation equipment: sterile operation room or ultra-clean workbench, sterile clothes, masks, caps, disinfection shoes, Alcohol lamp, etc. 5.1.1 Aseptic room is divided into aseptic operation room and buffer room. In the buffer room, there should be a hand basin, hand dryer, aseptic gowns and hooks, slippers and so on. Aseptic operation room should have air sterilization filtration laminar flow device, local cleanliness 100 ultra-clean bench. Buffer room and operation room are set up to achieve air disinfection of ultraviolet light and lighting, operation room or bench should maintain positive air pressure. 5.1.2 sterile room should be weekly and each time before the operation of 0.1% Xinjieermei or 2% cresol liquid wipe the operation table and possible contamination of the dead space, open the sterile air filters and ultraviolet light sterilization for 1 hour. At the end of each operation, the same 2% cresol or 0.1% Neosporin solution to wipe the working table, with ultraviolet light sterilization for half an hour. 5.1.3 Check the degree of sterility of the sterile room: aseptic room in the disinfection treatment, aseptic test and the operation process need to check the number of bacteria colonies in the air. Take double plates of 90mm diameter, light the alcohol lamp in the inoculation room, next to the alcohol lamp, with aseptic operation, half open the double plates into the dissolved nutrient agar medium of about 20ml, made of plates: pre-culture at 35-37 ℃ for 48 hours, after proving aseptic, will be brought into the aseptic operation room in aseptic manner of three plates in the clean area of the left, center, right each put one; open the disk cover buckle, the plate in the air exposure for 30 minutes, then cover the lid, put the plate in a sterile operation room, and put the plate in the air. After 30 minutes, put the lid on, incubate at 35-37℃ for 48 hours, take out and check, the total number of colonies growing on the 3 plates shall not be more than 10. Aseptic operating table or ultra-clean bench should regularly ask the relevant departments to test its cleanliness, should reach level 100 (generally with a dust particle counter), the number of particles of dust particle size ≤ 5μm shall not exceed 3.5 / liter; air flow should be controlled at 0.75-1.0m3 / s; bacterial colonies on average & lt; 1, according to the sterile condition of the regular replacement of filters.5.1.4 Aseptic room Inside should be prepared with a glass jar containing 5% cresol for disinfection, alcohol lamp, matches, tweezers, 75% alcohol cotton and slippers, etc. 5.2 Instruments and utensils: 5.2.1 Vacuum pumps, thermostatic incubator, biological microscope, pallet balance (accuracy 0.1g), pumping bottle (500ml), triangular bottles (100, 500 ml), pipettes (1, 10 ml), syringes (required specifications) Loosely stuffed into a small amount of raw cotton, and then put into the stainless steel sterilization cylinder. 5.2.2.2 test tubes in the mouth of the tube stuffed with gauze cotton plugs. 5.2.2.3 sterile clothes, pants, hats, masks will be washed clothes, pants, hats, masks supporting the bag into a cloth bag, tighten the mouth of the bag, and then wrapped in kraft paper. 5.2.2.4 syringes washed and cleaned syringes and syringe needles assembled into a gauze-padded container with a cover 5.2.2.4 Syringe clean syringes and injection needles are assembled and put into a container with a lid padded with gauze (rice Page 3/***7 box), put in one layer, covered with gauze, and then covered with the lid of the container, wrapped with kraft paper.5.2.2.5 Filter After the test is qualified, the microporous filter membrane is first soaked and moistened with water, and then taken out and fixed in the bacterial filter on the filter plate, and a high-temperature-resistant washer is used to gasket the filter under the filter plate, and then put on a good filter. Before sterilization of the filter screw do not screw too tightly, the upper mouth of the filter with 8 layers of gauze and kraft paper wrapping, properly loaded into the container, and then the container will hold the filter lid, wrapped in kraft paper. 5.2.3 Sterilization of utensils: will be wrapped in good utensils, in the 121 ± 0.5 ° C steam sterilizer cabinet sterilization for 30 minutes, the items out of the Do not immediately put in a cold place, so as to avoid rapid cooling of the items within the steam condensation caused by the sterilization of negative pressure, easy to cause bacterial infection, and the sterilization of the bacteria. Negative pressure, easy to cause bacterial infection, should be placed in a warm box or or heating and drying. 5.3 culture medium, reagents: 5.3.1 general use of commercial dry medium, ready to use in accordance with the instructions for the preparation of the medium, it should be noted that the culture medium pH should be in accordance with the provisions of, otherwise it must be corrected, sterilized for use. Before use, according to the requirements of the sterilization of bacterial culture medium shall be 30-35 ℃ cultured 48 hours, fungal culture medium shall be 20-25 ℃ cultured for 72 hours, to prove that the growth of sterile before use. Prepared aerobic and anaerobic culture medium should be used up within half a month, before inoculation of the test material, the color of the oxidized layer of the culture medium indicator shall not exceed the depth of the medium by about 1/5, otherwise it shall be boiled and heated in a water bath and heated only once.5.3.2 Gram's iodine solution: firstly, use 3-5ml of distilled water to dissolve 2.0g of potassium iodide, then add 1.0g of iodine tablets, stir to dissolve and dilute it with distilled water to 300ml, shake well. Place in a closed brown bottle for spare. 5.3.3 Crystalline violet staining solution: dissolve 1.0g of crystalline violet in 20ml of 95% ethanol, mix with 80ml of 1% ammonium oxalate solution, and let it stand for 48 hours. This solution is stable, in a closed brown bottle can be stored for several months.5.3.4 Sha Huang staining solution: 0.2g of Sha Huang dissolved in 10ml of 95% ethanol, to be completely dissolved and then add distilled water to 100ml.5.3.5 Saline: weighing 9g of sodium chloride, add water to dissolve 1,000ml, divided into portions and then sterilized at 121 ± 0.5 ℃ in wet heat for 30 minutes. For use as a diluent. Page 4/***7 5.3.6 75% ethanol: measure 75ml of anhydrous ethanol, dilute with water to 100ml, shake well, that is, 5.3.7 0.1% Neosporin: measure 5% Neosporin 20ml, dilute with water to about 1000ml, shake well, that is, 5.4 Medium sensitivity check: 5.4.1 Newly purchased dry medium or use different grades of raw materials of the The quality of fresh culture medium should meet the requirements of sensitivity check. (1) Take fresh cultures of Garcinia micrococcus [Micrococcus luteus CMCC (B) 28001] nutrient agar slant and Candida albicans [Candida albicans CMCC (F) 98001] fungal culture medium agar slant, respectively, and make a uniform bacterial suspension with 0.9% sterile sodium chloride solution; take Clostridium sporogenes [Clostridium sporogenes], and make a uniform bacterial suspension; take the culture medium of Clostridium sporogenes [Clostridium sporogenes] and Clostridium sporogenes [Clostridium sporogenes]. Clostridium sporogenes CMCC (B) 64941] fresh cultures of agar-free aerobic and anaerobic medium were sucked into a sterilized centrifuge tube with a sterilized capillary tube, centrifuged, the supernatant was discarded, and the precipitated organisms were made into a homogeneous bacterial suspension with 0.9% sterile sodium chloride solution. Then diluted in a series of 10 times, made to contain 10-100 bacteria in 1ml and counted. (2) Take the fresh cultures of aerobic and anaerobic medium of Garcinia micrococcus and Clostridium perfringens, and the fresh cultures of fungal medium agar slant of Candida albicans, and take the inoculation into the mold medium, and then make a 10-fold dilution with 0.9% sterile sodium chloride solution at 20-25℃, and make it into a 1 ml containing 10-100 bacteria and count them. Inoculate 1 ml each of the above dilutions of (1) or (2) of Micrococcus garciniae into 3 tubes of aerobic and anaerobic medium with a loading of 9 ml per tube, 1 ml each of the above dilutions of (1) or (2) of Clostridium perfringens into 3 tubes of aerobic and anaerobic medium with a loading of 12 ml per tube, 1 ml each of the above dilutions of (1) or (2) of Candida albicans into 3 tubes of fungi medium with a loading of 9 ml per tube, and 1 ml each of the above dilutions of (1) or (2) of Candida albicans into 3 tubes of fungi medium with a loading of 12 ml per tube. Inoculate 3 tubes of 9 ml of fungal medium, use the uninoculated medium as a control, incubate at the specified temperature for 5 days and record the results day by day. The result is determined as follows: no less than 2 tubes of culture medium after inoculation of each strain of bacteria shall show growth, i.e. the sensitivity check of the medium meets the requirements.5.4.2 The prepared culture medium shall be stored in a cool and dark place, generally not more than 2 weeks, and the bacterial and mold culture medium shall be cultured at 30-35℃ and 20-25℃ for not less than 48 hours and 72 hours respectively before use, and shall be used only after the proof of sterility of growth.5.4.3 Aerobic bacteria, anaerobic bacteria culture medium in the test tube loading height shall not be less than 7Cm, its indicator oxidized layer shall not exceed the depth of the culture medium 1 / 5, otherwise it must be boiled by a water bath for 10 minutes, but only heat once. Page 5/***7 aseptic test at the end of the incubation time, the indicator oxidized layer should not exceed 1/2 of the depth of the medium. 5.5 Preparation of the control solution: 5.5.1 test strains, strain recovery, strain inoculation and preservation should be operated in accordance with the "microbiological assay of antibiotics," under the standard operating procedures. 5.5.2 Staphylococcus aureus bacterial liquid with an inoculating ring to take a few fresh cultures of Staphylococcus aureus [Staphy-lococcus aureus CMCC (B) 26003] of the nutrient agar slant, inoculated into the nutrient broth medium, cultured at 30-35 ℃ for 16-18 hours, diluted with sterilized physiological saline to 1:1065. 5.5.3 Clostridium sporotrichum Fungal solution with an inoculating ring to take Clostridium spp. [CMCC (B) 64941] aerobic, anaerobic medium fresh culture 1 platinum ear, inoculated into the same medium, incubated at 30-35 ℃ for 18-24 hours, then diluted with sterilized saline to 1:106.5.5.4 Candida albicans Fungal solution with an inoculating ring to take Candida albicans [CMCC (F) 98001] fungi agar medium slant fresh culture 1 platinum ear, inoculated into the fungal medium, incubated at 20-25 ℃ for 24 hours, and then diluted to 1:105 with sterilized saline. 5.5.5 The fungal solution prepared above is usually used on the same day. 5.6 Operational points of entering the aseptic room:5.6.1 According to the test procedure, move the utensils and articles into the buffer room, turn on the ultraviolet lamps and air filtration devices and make them work for more than 1 hour. 5.6.1 According to the test procedure, move the utensil items into the buffer room, turn on the UV lamp and air filtration device and make It works for more than 1 hour.5.6.2 Operators brush their hands with soap and water, turn off the ultraviolet light in the buffer room, enter into the buffer room, close the door of the first floor, wash their hands with 2% Lysol or 0.1% Neosporin solution, dry them with sterilized towel, and put on the sterile gowns, hats, masks, and sterilized shoes.5.6.3 Turn off the ultraviolet light in the aseptic room, enter into the door of the second floor, and move the utensils to the aseptic room, and close the door of the aseptic room.5.6.2 Operators should not use the sterile gowns, caps, masks and sterilized shoes, but use the sterile gowns, caps, masks and sterile shoes to clean the hands. The sterile room door is closed.5.6.4 Anyone entering the sterile room should not go out to retrieve items; therefore, items used in each test must be planned and spares must be available. From the time of entry to the first door up to the sterile room, a 2% Lysol or 0.1 % Neosporin solution should be sprayed as the operator enters.5.7 Inspection Methods: 5.7.1 Aseptic inspection methods include: direct inoculation and membrane filtration. The former applies to non-antimicrobial effect of the test material; the latter applies to have an antimicrobial effect or large-volume test material. 5.7.2 operation, should be soaked or wiped with 0.1% Neosporin after the surface of the container to take the aseptic method of Page 6 / **** 7 contents. 5.7.3 Where aseptic examination, should be taken in the same way as the corresponding solvents and dilutions, as a negative control.5.7.4 Preparation of the test material : Remove the syringe with sterilized tweezers, next to the flame will be inserted into the needle core and needle, cover for the rubber stopper, the syringe has been sterilized in the rubber stopper in the center of the position of the prick to absorb the liquid. According to the provisions of the test material to be diluted or inactivated, can be directly or the bottle of test liquid drawn to the sterilization of glass containers for inactivation and dilution to the required volume and concentration; extract the contents of the bottle of liquid, the test material should be inverted and so that the needle in the liquid under the surface of the liquid.5.7.5 Direct inoculation method: according to the prescribed amount of test material, aseptic operation will be inoculated in the test material were inoculated in the aerobic, anaerobic bacteria medium 6 tubes, of which 1 tube inoculated with Staphylococcus aureus liquid, and 1 tube inoculated with Staphylococcus aureus liquid. The tube is inoculated with 1ml of Staphylococcus aureus liquid as positive control, and the other is inoculated in 5 tubes of fungal medium. Shake gently to mix the test material with the medium. Aerobic and anaerobic medium tubes are placed at 30-35℃, and fungal medium tubes are placed at 20-25℃ for 7 days. During the incubation period should be observed day by day and recorded whether there is bacterial growth. Positive control tube should have bacterial growth within 24 hours, such as after adding the test material, the medium appears turbid, after 7 days of culture, can not judge from the appearance of microbial growth, can take the culture solution appropriate amount of transfer to the same kind of fresh medium or slant medium to continue to cultivate, the bacterial culture for 2 days, fungal culture for 3 days, to observe whether there is no bacterial growth of turbid or slant again, or with inoculation ring to take the culture solution smear 5.7.6 Membrane filtration: connect the microporous membrane filtration device, filtration bottle, exhaust pipe and vacuum pump, the vacuum pump can be placed outside the sterile room. Take the specified amount of test material, according to the specified method of treatment, add 0.9% sterile sodium chloride solution 100ml, mixed, through the membrane filter equipped with a pore size of not more than 0.45μm, decompression and drying, with 0.9% sterile sodium chloride solution rinse the membrane for 3 times, at least 100ml each time, take out the membrane divided into 3 equal portions, respectively, added to the above two kinds of culture medium, according to the specified temperature and time. Cultivate according to the specified temperature and time. Positive control tube should be added according to the characteristics of the test material 1ml of the corresponding control bacteria (antibacterial drugs, Staphylococcus aureus as the control bacteria; anaerobic drugs, Clostridium perfringens as the control bacteria; antifungal drugs, Candida albicans as the control bacteria). Positive control tube bacteria should be cultured for 24-48 hours, fungi should be cultured for 24-72 hours with bacterial growth. 5.8 Judgement of the results: Page 7/***7 When the positive control tube is turbid and there is indeed bacterial growth, the negative control tube is negative, according to the observation of the results of the judgment: such as aerobic bacteria, anaerobic bacteria and fungal culture medium tube are clarified or turbid, but proved to be not a bacterial growth, should be judged as a qualified test; such as aerobic bacteria, anaerobic bacteria and fungal culture medium tube is clarified or turbid but not bacterial growth, should be judged as a qualified test. If any one of the tubes of aerobic, anaerobic and fungal medium is turbid and confirmed to have bacterial growth, two times the amount of test material should be taken again and retested in accordance with the law, and there should be no bacterial growth in other tubes except for the positive control tube, otherwise the test material should be judged to be unqualified.6 Training 6.1 Target: laboratory technician.6.2 Training time: two hours.7 Records Record name Retention department Retention time Aseptic test record Quality Supervision Section One year after the expiration date of drugs QF-01-007-00 Aseptic test record Product name: Batch No.: Specification: Date of test: Year Month Day Medium name aerobic, anaerobic bacterial medium Fungal medium Cultivation Cultivation Temperature 30-35 ℃ 20-25 ℃ Cultivation time Tube No. 1234512345 Cultivation days and results1234567Conclusion Remarks Reviewer: Inspection: