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Sterile room standardization procedures and acceptance specifications
1. Purpose
This procedure aims to provide a standardized procedure for aseptic operation and protection of sterile rooms.
2. Scope of application
Bioassay laboratory
3. Responsible person
QC supervisor bioassay personnel
4. Definition
5. Safety precautions
Strictly perform aseptic operation to prevent microbial contamination; operators should turn off the ultraviolet light before entering the sterile room.
6. Procedures
6.1. The sterile room should be equipped with a sterile operation room and a buffer room. The cleanliness of the sterile operation room should reach Class 10000, the indoor temperature should be maintained at 20-24℃, the humidity should be maintained at 45-60%, and the cleanliness of the Clean bench should reach Class 100.
6.2. The sterile room should be kept clean, and it is strictly forbidden to pile up debris to prevent contamination.
6.3. Strictly prevent all sterilization equipment and culture media from being contaminated, and those that have been contaminated should be stopped from use.
6.4. The sterile room should be equipped with disinfectants of working concentration, such as 5% cresol solution, 70% alcohol, 0.1% chlorhexidine solution, etc.
6.5. The sterile room should be sterilized and cleaned regularly with appropriate disinfectants to ensure that the cleanliness of the sterile room meets the standards.
6.6. All items such as instruments, equipment, plates, etc. that need to be brought into the sterile room for use should be tightly wrapped and sterilized by appropriate methods.
6.7. Before entering the sterile room, the staff must wash their hands with soap or disinfectant, and then change into special work clothes, shoes, hats, masks and gloves in the buffer (or wipe their hands again with 70% ethanol) before entering the sterile room for operation.
6.8. Before using the sterile room, the ultraviolet light in the sterile room must be turned on for irradiation and sterilization for more than 30 minutes, and the clean bench must be turned on for blowing at the same time. After the operation is completed, the sterile room should be cleaned in time and then irradiated and sterilized with ultraviolet light for 20 minutes.
6.9. Before inspection, the outer packaging of the test sample should be kept intact and should not be opened to prevent contamination. Before inspection, the outer surface should be disinfected with 70% alcohol cotton balls.
6.10. During each operation, a negative control should be performed to check the reliability of aseptic operation.
6.11. When sucking bacterial waves, you must use an ear suction bulb to suck, and never touch the straw directly with your mouth.
6.12. Before and after each use, the inoculation needle must be sterilized by flame burning. After cooling, the culture can be inoculated.
6.13. The straws, test tubes, culture dishes and other utensils with bacterial liquid should be immersed in a disinfection bucket containing 5% Lysol solution for disinfection, and then taken out and rinsed after 24 hours.
6.14. If bacterial liquid is spilled on the table or the ground, it should be immediately poured with 5% carbolic acid solution or 3% Lysol on the contaminated area for at least 30 minutes before processing. When work clothes and hats are contaminated by bacterial liquid, they should be taken off immediately and washed after high-pressure steam sterilization.
6.15. All items with live bacteria must be disinfected before being rinsed under the faucet. It is strictly forbidden to pollute the sewer.
6.16. The number of colonies in the sterile room should be checked every month. With the clean bench open, take several sterile culture dishes with an inner diameter of 90mm, and aseptically inject about 15ml of nutrient agar culture medium that has been melted and cooled to about 45℃.
After solidification, invert it in a 30~35℃ incubator for 48 hours.
After proving sterility, take 3~5 plates and place them on the left, middle, right, etc. of the working position.
After opening the cover and exposing for 30 minutes, invert it in a 30~35℃ incubator for 48 hours, and take it out for inspection. The average number of colonies on the plate in the 100-level clean area shall not exceed 1 colony, and the average number of colonies in the 10000-level clean room shall not exceed 3 colonies. If it exceeds the limit, the sterile room should be thoroughly disinfected until repeated inspections meet the requirements.
7. Reference
Refer to the chapter (sterility inspection method) in "Drug Hygiene Inspection Methods" and "China Drug Inspection Standard Operation Specifications" and the People's Republic of China Pharmaceutical Industry Standard VY/T0188.6-1995 "Drug Inspection Operation procedures"
8. Distribution Department
Quality Management Department
Aseptic Room Technical Instructions
To maintain an aseptic environment for studying microorganisms, it is crucial to prevent contamination from external sources, known as contaminated bacteria. Effective aseptic techniques involve thorough sterilization and measures to contain both pathogenic and genetically engineered microorganisms within experimental settings.
Aseptic rooms must have flat, easily cleaned floors and walls, and the workbench should be horizontal. Ultraviolet lamps are placed between the sterile room and buffer areas, and personnel must wear sterilized clothing and masks. Clean benches are commonly used in laboratories, utilizing laminar airflow to remove dust and microorganisms.
In challenging conditions, wooden sterile boxes can serve as portable alternatives to clean benches. These boxes feature two front openings, covered when not in use, and a glass top for visibility. An internal ultraviolet lamp helps maintain sterility, while instruments can be accessed through a side door.
Application
Aseptic operation technology currently plays a pivotal role not only in microbiological research and application, but also in many biotechnologies. For example, transgenic technology, monoclonal antibody technology, etc.
Standard operating procedures for clean room sedimentation bacteria testing
1. Purpose
To establish standard operating procedures for testing sedimentation bacteria in clean rooms to ensure that drugs are produced within the specified clean level.
2. Scope
monitoring of sedimentation bacteria in clean rooms.
3. Basis
National standard GB/T16294-1996.
4. Responsibilities
QA cleanliness monitoring personnel and microbiological inspection personnel are responsible for the implementation of this system.
5. Content
5.1 Clean room
A room or area that requires environmental control for dust particles and microbial contamination:
5.2 Clean workbench
A workbench or a similar enclosed enclosure work area. Its characteristics are that it can supply filtered air or gas, such as vertical laminar clean hood, horizontal laminar flow hood, vertical laminar clean workbench, horizontal laminar clean workbench, self-cleaning device, etc.
5.3 Cleanliness
The permissible statistical number of suspended particles greater than or equal to a certain particle size in a unit volume of air in a clean environment.
5.4 Colony
A bacterial colony formed by the reproduction of one or several bacteria after bacterial culture, referred to as CFU. Usually expressed in number.
5.5 Test method
5.5.1 Method overview: This test method uses the sedimentation method, that is, the biological particles in the air are collected on the culture medium plate through the principle of natural sedimentation, and after more than 48 hours of cultivation, they are allowed to reproduce to a visible colony number under suitable conditions to assess the number of live microorganisms in the clean environment, and thus the cleanliness of the clean room is assessed.
5.5.2 Instruments and equipment used
5.5.2.1 High pressure sterilizer: When using, refer to the document GZ-ZL-SOP-066-00 for operation
5.5.2.2 Constant temperature incubator: The thermometer of the incubator must be calibrated regularly.
5.5.2.3 Culture dish: Generally, a medium 90mmx15mm borosilicate glass culture dish is used. Before use, place the culture dish in 121℃ moist heat sterilization for 20 minutes.
5.5.2.4 Culture medium: ordinary nutrient agar culture medium. Heat the culture medium to melt, cool to about 45℃, and inject the culture medium into the culture dish under aseptic operation conditions, about 15ml per dish.
After the agar culture medium is dissolved, place the culture dish in a 30~35℃ constant temperature incubator for several hours. If there is no colony growth on the culture dish, it can be used for sampling. The prepared culture dish should be stored in an environment of 2~8℃.
5.5.3 Test steps
5.5.3.1 Sampling method: Place the prepared culture dish at the predetermined sampling point, open the culture dish cover, expose the culture medium surface for 0.5 hours, and then cover the culture dish and turn it upside down.
5.5.3.2 Culture: After all sampling is completed, place the culture dish upside down in a constant temperature incubator for culture. Culture at 30~35℃ for no less than 48 hours. Each batch of culture medium should have a control test to check whether the culture medium itself is contaminated. Three culture dishes can be selected for control culture in each batch.
5.5.3.3 Counting of 10,000 colonies: Count directly with the naked eye, and then check with a 5-10x magnifying glass to see if there are any omissions. If there are 2 or more colonies on the culture dish, count as 2 or more colonies when they can be distinguished.
5.6 Precautions
5.6.1 The test equipment should be sterilized to ensure the reliability and correctness of the test.
5.6.2 Take all measures to prevent human contamination of the sample.
5.6.3 Keep detailed records of the culture medium, culture conditions and other parameters.
5.6.4 Due to the wide variety of bacteria and their great differences, when counting, generally use transmitted light to carefully observe the back or front of the culture dish. Do not miss the colonies growing on the edge of the culture dish, and pay attention to the difference between bacterial colonies and culture medium sediments. Use a microscope to monitor if necessary.
5.6.5 The quality of each culture dish should be carefully checked before sampling. If it is found to be deteriorated, damaged or contaminated, it should be discarded.
5.7 Test rules
5.7.1 Test status
5.7.1.1 Before the sedimentation bacteria test: the temperature and humidity of the clean room (area) to be tested must meet the specified requirements, and the static pressure difference must be controlled within the specified value. The clean room (area) to be tested has been disinfected.
5.7.1.2 There are two test states: static and dynamic, and the test state shall be indicated in the report.
5.7.1.3 Testers: Testers must wear work clothes that meet the environmental cleanliness level. During static testing, there shall be no more than 2 testers in the room.
5.7.2 Test time
5.7.2.1 For unidirectional flow, such as in a Class 100 Clean room and a laminar flow workbench, the test should start after the purification air conditioning system has been operating normally for at least 10 minutes.
5.7.2.2 For non-unidirectional flow, such as clean rooms of Class 10,000 and above, the test should start after the Clean air conditioning system has been operating normally for at least 30 minutes.
5.8 Settling bacteria count
5.8.1 Minimum number of sampling points
While meeting the minimum number of measurement points, the minimum number of culture dishes should also be met.
5.8.2 Arrangement of sampling points: The test point position in the working area is about 0.8~1.5m above the ground (slightly higher than the working surface). Measuring points can be added at key equipment or key work activity ranges. The arrangement of sampling points should be uniform to avoid sampling points being too concentrated in a certain local area and too sparse in a certain local area.
5.8.3 Records: The room temperature, relative humidity, pressure difference, test status and test data should be recorded in the test report.
5.8.4 Result calculation: The number of colonies in each culture dish is obtained by counting method. The calculation of the average colony count is as follows:
m1+m2+......+mn
Average colony count m=n
Where: m is the average colony count;
m1 is the colony count of culture dish No. 1
m2 is the colony count of culture dish No. 2
mn is the colony count of culture dish No. n
n is the total number of culture dishes.
5.8.5 Result evaluation: Use the average colony count to judge the microorganisms in the air of the clean room.
5.8.6 The average colony count in the clean room (area) must be lower than the selected evaluation standard.
5.8.7 If the average colony count of A Clean Room (area) exceeds the evaluation standard, the area must be disinfected first, and then retested twice, and the test results must be qualified.
Cleanliness level 100 level 10000 level 100000 level 300000 level
Standard (average) 1/dish 3/dish 10/dish 15/dish
Main references: National standard GBTT16294-1996.
