A laboratory built in accordance with the BSL-3 standard, also known as a biosafety laboratory. The laboratory needs to be isolated from the general area of ​​the building. The specific standard microbiological operations, special operations, safety equipment, and laboratory facilities are as follows.
3.1 standard operation
After completing the infectious material operation, disinfect the gloves and remove the gloves and wash your hands before leaving the laboratory. It is forbidden to eat, drink or smoke in the facility, and it is not allowed to touch contact lenses and use cosmetics. People wearing contact lenses should also wear goggles or face shields. Food can only be stored outside the work area. Do not use the mouth to draw the test liquid. Use a special pipette. Care must be taken in all operations to reduce and avoid aerosols. The laboratory hygiene should be cleaned at least once a day, and the work surface should be disinfected at any time after work. The infectious materials should be disinfected immediately when they spill out and spill. All cultures, stocks, and other daily waste are effectively sterilized using an autoclave prior to disposal. Materials that need to be handled outside the laboratory should be placed in a tight, leak-tight container, sealed and passed out of the laboratory. Laboratory waste should be sterilized and packaged before being sent to the disposal site to avoid environmental pollution. The bacteria and poisons operating in BSL-3 must be kept by two people and kept in a safe and reliable facility. Before use, they should go through the approval procedures, explain the dosage, and register in detail. The two people can be taken out at the same time. The test should have detailed use and destruction records. Insect and rodent control should be carried out in accordance with other relevant regulations.
3.2 special operations
Develop safety rules
The person in charge of the laboratory shall formulate special and comprehensive biosafety rules and specific operational procedures of the laboratory according to the actual situation to supplement and refine the operational requirements of this specification and report to the Biosafety Committee for approval. Staff members must understand the rules and implement them carefully.
Biohazard symbol
Mark the international generic biohazard symbol on the door of the laboratory entrance. The laboratory door is marked with the experimental microbial species, the list of laboratory leaders and the telephone number, indicating the special requirements for entering the laboratory, such as the need for immunization, wearing protective masks or other personal protective equipment. During the use of the laboratory, no one is allowed to visit. If the visit must be approved and entered when the individual conditions and protections are met.
Biohazard warning
When infectious materials or infected animals are placed in the laboratory or physical protective equipment during the experiment, the laboratory doors must be kept tightly closed, and unrelated personnel are not allowed to enter. The door should be marked with a hazard warning sign. If the red card or text is used to indicate the state of the experiment, it is forbidden to enter or approach.
Conditions for entering the laboratory
The person in charge of the laboratory shall designate, control or prohibit the laboratory and supporting personnel entering the laboratory. Minors are not allowed to enter the laboratory. Laboratory staff with an increased probability of infection or serious consequences of infection are not allowed to enter the laboratory. Only those who understand the potential biohazard and special requirements of the laboratory and who comply with the relevant regulations can enter the laboratory. Animals and plants and other items not related to work are not allowed to be brought into the laboratory.
Staff training
Regular and unscheduled professional training in biosafety protection related to work is required for laboratory staff and support staff. The laboratory personnel are required to undergo specialized biological professional training and biosafety training, and are guided by experienced experts or under the supervision of the Biosafety Committee. The evaluation and prevention of aerosol exposure hazards must be learned. Before the infectious work in the BSL-3 laboratory, the person in charge of the laboratory must ensure and prove that all the staff are proficient in microbiological standard operation and special operation, and are proficient in the special operation and operation technology of the equipment and facilities of the laboratory. Includes skills to operate pathogenic factors and cell culture, or special training for laboratory leaders, or include expert and safety committee guidance under the guidance of a safety microbiologist. Avoid aerosol exposure: The handling of all infectious materials should not be directly exposed to the air. They should not be carried out on open countertops and in open containers. They should be carried out in biosafety cabinets or other physical protective equipment. The need to protect the human body and samples can be performed in an indoor discharge type 2A biosafety cabinet. The operation of protecting only the human body from protecting the sample can be carried out in a Class I biosafety cabinet. If operating with radioactive or chemical hazards, it should be in Type 2B2 Biosafety Cabinet. It is forbidden to use a clean bench.
Avoid sharp weapon infections
For potentially contaminated devices, including needles, syringes, blades, glass slides, straws, capillary straws, and scalpels, highly effective precautions must be taken frequently to prevent transdermal laboratory infections. Work in the BSL-3 laboratory and try not to use needles, syringes and other sharp devices. Use only when necessary, such as injection of a parenchymal organ, venous incision, or taking liquid from an animal and a bottle (seal cap). Try to replace the glass with a plastic product. When injecting and extracting infectious materials, use a disposable syringe (the needle is integral with the syringe). Use the used needle to avoid unnecessary operations before disinfection, such as non-bending, breaking, and breakage. Do not cover the original needle cap directly with your hand; carefully place it in a convenient and non-piercing treatment tool. The container is then autoclaved. Broken glass cannot be handled directly by hand and must be removed mechanically, such as brushes, clips and tweezers.
Pollution removal and disinfection
After the operation of the infectious material is completed, the laboratory equipment and the work surface are disinfected by using an effective disinfectant, especially the overflow of the infectious material, splashing other pollution, and timely disinfection. Disinfection of spilled infectious materials is handled and removed by a suitable professional or by other trained and experienced workers using high concentrations of infectious agents. All waste must be autoclaved prior to disposal. All potential laboratory contamination (eg, gloves, overalls, etc.) must be disinfected prior to disposal or disposal. Instruments that require repair and maintenance should be disinfected before shipment.
Storage and transportation of infectious samples
All infectious samples such as cultures, tissue materials and body fluid samples should be placed in a non-leakage container during storage, handling and transportation. The outer surface of the container should be thoroughly disinfected and the packaging should have obvious and firm marks.
Monitoring of pathogen traces
Background serum samples from all laboratory staff and other relevant personnel were collected for pathogen trace detection. Regular and irregular collection of serum samples for specific testing based on the function of the pathogen and facility being operated or in actual events.
Medical supervision and health care
Medical supervision and care are provided to workers during the work of the BSL-3 laboratory. For laboratory-operated pathogens, the staff is subject to appropriate testing or immunization (eg rabies vaccine, TB skin test).
Treatment of exposure accidents
When there is continuous positive pressure in the biosafety cabinet or laboratory, the indoor personnel should immediately stop the operation and put on a protective mask to take measures to restore the negative pressure. If the negative pressure cannot be recovered and maintained in time, the experiment should be stopped and exited as early as possible. In the event of such an accident or other accidents and pollution with a potential risk of contagious exposure, the party shall immediately report to the person in charge of the laboratory, wait for instructions, and report to the National Veterinary Laboratory Biosafety Management Committee in addition to emergency measures. The responsible person and the party shall urgently and scientifically handle the accident. After the event, the parties and the responsible person should provide a realistic medical hazard evaluation, medical supervision and preventive treatment. The person in charge of the laboratory shall investigate and publish the process of the incident, write a written report to the National Veterinary Laboratory Biosafety Management Committee and copy the laboratory safety committee and keep the backup.
3.3 Safety equipment (primary protective barrier)
Protective clothing
In the laboratory, workers should wear protective laboratory clothing, such as long clothing, short suits, or work clothes with chest protection. After disinfection, if there is obvious pollution, it should be replaced in time and treated as waste. Work clothes should not be worn outside the laboratory.
protective gloves
Gloves must be worn when handling infectious materials, infected animals and contaminated instruments. It is better to wear double layers and wear protective gloves that are not easily damaged if necessary. Dispose of the gloves before use, and disposable gloves should not be reused.
Biological safety cabinet
The operation of infectious materials, such as the anatomy of infected animals, tissue culture, chicken embryo inoculation, and collection of animal body fluids, should be carried out in a biological safety cabinet of Class II or higher. Centrifugation, comminution, agitation, etc., which cannot be performed in a Class II biosafety cabinet, can be performed in larger or special Class I biosafety cabinets.
Other physical protection
When the operation cannot be carried out in the biosafety cabinet, personal protection (specific requirements for similar protective equipment of Class III or higher) and other physical protective equipment (centrifuge safety helmet, or sealed centrifuge rotor) are used together.
Facial protection
Contaminated areas and semi-contaminated areas should be equipped with protective masks for emergency use, and mask protection should be provided when there are infected animals in the room. Establish emergency protection work points.
Emergency protective equipment
Spare protective masks, equipment for washing eyes and medicines in contaminated areas or semi-polluted areas are readily available.
3.4 Laboratory facilities (secondary protective barrier)
The operation of the original microorganisms in the BSL-3 Biosafety Laboratory is carried out in Class II and above (including Class II) biosafety cabinets. The secondary barrier standards are as follows:
Building structure and layout
The building has more than seven earthquake resistance, anti-rat, insect-proof and anti-theft. The net height of the laboratory should be above 2.6 meters, and the net height of the pipeline should not be less than 2.0 meters. The laboratory in the building should be separated from the public area where the activity is not restricted. Safety doors should be installed and door locks should be installed to prohibit unrelated personnel from entering. The access to the facility is provided with a double door with a door closer, followed by a dressing room, which is divided into a further room (cleaning area) and a second room (semi-contaminated area), and the second room is followed by a rear room or buffer room ( Semi-contaminated area), the doors that enter and exit the buffer chamber should be automatically interlocked. If multiple laboratories share a common corridor (or buffer room), then each laboratory should go through a chain of airlock (lock) doors. The laboratory should have a safe passage and emergency exit and be clearly marked. A transfer window must be provided between the semi-contaminated area and the clean area. The auxiliary area such as the washing room and the machine room should be a clean area, but the distance from the laboratory should be shortened as much as possible to facilitate work. A closed observation window can be provided in the experimental room.
Airtightness and inner surface
All facilities and equipment have no burrs on the outside, no sharp edges and corners, minimize the horizontal surface area, and are easy to clean and disinfect. The holes through which the various pipes pass must be sealed. The surface of the wall and ceiling should be smooth, not glare, dusty, free from chemicals and common disinfectants, no water seepage, no condensation. The surface of the ground should be integrated, non-slip, wear-resistant, corrosion-resistant, non-reflective, dust-free, and water-tight. If it can be divided according to the pollution area, the color difference is better. The work surface is not water permeable and resistant to moderate heat, organic solvents, acids, alkalis and common disinfectants. The necessary table and chair cabinets in the laboratory are designed in advance to ensure safe placement and use. There is a certain space for each other to facilitate cleanliness, convenient surface disinfection and corrosion resistance.
Sterilization facility
A double-head type autoclave must be installed between the semi-contaminated area and the scrubbing unit. The two doors of the sterilizer should be interlocked with each other, and the sterilizer should meet the biosafety secondary sterilization requirements. UV lamps can be installed in contaminated areas, semi-contaminated areas or in transfer windows. Manual or automatic disinfection equipment (such as disinfectant sprayer, ozone sterilizer) should be prepared indoors and adequate disinfectant should be provided. All wastes in the laboratory should be classified and stored in reliable containers. Disinfection (high pressure, chemical, incineration, other treatment) should be carried out in the facility. The appropriate method for disinfection of the instrument, such as transfer ozone disinfection. Cabinets, ethylene oxide sterilization bags, etc., if the waste needs to be transferred to the laboratory, it should be disinfected and put into a sealed container and packaging.
Purifying air conditioner
The polluted area and semi-polluted area of ​​the laboratory adopt a negative pressure one-way flow fresh air purification air conditioning system. Heating and split air conditioning are not allowed in contaminated areas and semi-polluted areas, and electric fans are not available. The temperature is 230C±20C and the relative humidity is 40%-70%. Indoor noise does not exceed 60 decibels. The direction of the airflow is always guaranteed to flow from the clean zone to the contaminated zone, from the low-pollution zone to the high-pollution zone. The air-conditioning system should be equipped with a pressure-independent device to ensure the system pressure balance, and the exhaust air should be equipped with an automatic switching system. In the event of an emergency, the air supply system should be turned off to maintain exhaust air and to ensure safe negative pressure in the laboratory. The gas supply needs to be filtered by HEPA. The vented gas must be vented through at least two levels of HEPA filtration and is not allowed to be recycled in any area.
The indoor cleanliness is higher than 10,000. The air supply port of the laboratory should be on the roof of one side, and the air outlet should be at the lower part of the opposite wall to minimize indoor airflow dead angle. Maintaining a unidirectional airflow, the flow pattern is more appropriate. A visual device is installed at the entrance of the laboratory to accurately indicate the direction of airflow into the laboratory. Class II biosafety cabinets are tested once a year. Type 2A exhaust can enter the room, and the exhaust of the 2B2 safety cabinet and the Class III safety cabinet is discharged through the laboratory exhaust system. If the Class III safety cabinet is equipped with secondary HEPA filtration and mobile, the airflow can also be exhausted indoors, but the exhaust port should be close to the indoor exhaust vent. If other equipment such as liquid disinfection transfer window, drug fumigation sterilizer, etc., must be filtered by HEPA and replaced as needed.
Water purification treatments are provided near the exit of each room with a non-manually switched sink. Contaminated areas, semi-contaminated areas and water pipelines that may be contaminated should be protected against backflow. If there is water, pool or floor drain, disinfection facilities should be installed. A water seal must be provided under the water and always filled with a disinfectant. The water seal should be filled with a HEPA filter. The contaminated sewage can only be discharged into the disinfection device, and then discharged to the public sewer. All wastewater that has not been discharged or that is not discharged must be collected and treated at high pressure. The sewage in the clean area can be discharged directly into the public sewer.
Contaminant and waste disposal
Items that may be contaminated and other wastes should be placed in a dedicated container that prevents contamination or can be sterilized for disinfection or autoclaving.
Laboratory monitoring system
The monitoring alarm points should be comprehensively set up in various states and facilities of the laboratory to form a complete laboratory safety alarm system. When the backup power supply is not dual-channel power supply, it should be equipped with a backup power supply. At the time of power failure, at least the air conditioning system, alarm bell, lighting, access control and bio-safety equipment can be guaranteed.
illumination
Lighting should be suitable for all activities in the room, no reflection, no glare, no impact on the line of sight. It is best to install the components of the luminaire in the ceiling or to reduce dust accumulation.
communication
There should be suitable communication facilities (telephone, fax, computer, etc.) inside and outside the laboratory for paperless operation. Acceptance and annual inspection of BSL-3 facilities and operations must be mandatory. The laboratory acceptance or annual inspection shall be carried out in accordance with the ISO10648 standard test method for sealing test. The detection pressure shall not be less than 250Pa, and the hourly leakage rate shall not exceed 10% to ensure the reliability of the maintenance structure. The function of the new facility must be checked and accepted to confirm that the design and operating parameters are in compliance with the requirements. A test confirmation is performed once a year after the operation.
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