How to build a biosafety laboratory

Biosafety has received more and more attention in the world, especially since the outbreak of atypical pneumonia (SARS) and highly pathogenic avian influenza. Domestic experts, scholars and ordinary people have recognized the grim situation facing China in the field of biosafety. Prior to this, China did not have a P4 laboratory, and the P3 laboratory was not standardized. This situation severely restricted China's research and application of biosafety. The Ministry of Health has issued an industry standard for biosafety laboratories, the General Guidelines for Biosafety in Microbiology and Biomedical Laboratories (WS233-2002), which is based primarily on the National Institutes of Health (NIH) and US Centers for Disease Control. The Center (CDC) standard is developed to guide the design, construction and use of biosafety laboratories at all levels. This article briefly introduces some of the main considerations for establishing a P3 laboratory that is commonly used in research such as SARS, and cites the characteristics and performance of specialized instruments and equipment that meet the requirements of the P3/P4 biosafety laboratory. The Control Center Laboratory Biosafety Level Standards (Levels 3 and 4) are attached for reference.
P3 laboratories are used for research on endogenous and exogenous sources (in case of inhalation of the source due to exposure, which can cause serious, potentially lethal diseases). In general, the laboratory should be professionally trained in dealing with pathogenic and potentially lethal sources and be supervised by qualified scientists who have experience working with the source. All procedures related to the operation of the source of infection should be carried out in a biosafety cabinet or other physical protection device, or by an experimenter wearing appropriate protective clothing and facilities. The laboratory must be specially designed and constructed. Here are a few specific requirements for the lab:
First of all, the location of the P3 laboratory is chosen to leave the walking area inside the building, and it is forbidden to enter the laboratory. After entering the aisle, at least two automatic doors are required to enter the laboratory. All doors should have a lock, and a locker room can be designed in the aisle to allow the experimenter to change the experimental protective suit. In addition to wearing experimental protective clothing, laboratory personnel must wear gloves when handling infectious sources, infected animals, and contaminated instruments. Respiratory and facial protection measures should also be taken when entering a room with infected animals.
In the laboratory, a fully automatic sink should be installed near the door. In the area where the source of the disease is treated, the surface of the wall, the floor and the ceiling should be smooth and waterproof, and the joints are sealed. It is corrosion-resistant to the common chemical reagents and disinfectants in the laboratory, and is easy to clean and disinfect. The floor is attention to integrity and non-slip, it is recommended to use a cover in the recess of the floor. The test table should be able to withstand the expected weight, and its surface material should be waterproof, heat resistant, resistant to organic solvents, acid and alkali and other disinfecting chemicals. Pay attention to the overall layout, the space between the laboratory table, the safety cabinet and the equipment should be easy to clean.
The laboratory should have a ducted exhaust system with wind directions from “clean areas” to “contaminated areas”. The exhausted air is not recycled to any other area within the building.
The external exhaust port should be away from the manned area and the air inlet, otherwise it needs to be filtered by HEPA. The experimenter must verify that the wind direction into the laboratory is correct. It is recommended to have a visual monitoring device at the entrance to the laboratory to indicate and confirm the wind direction of the incoming air. Consider installing an HVAC control system to prevent the laboratory from continuously boosting forward. At the same time, an audible alarm device should be installed to warn the experimenter that the HVAC system has a problem. And test the HEPA system at least annually to make sure it works.
All operations related to the source of infection, autopsy of infected animals, collection of tissues or fluids from infected animals or embryonic eggs should be performed in a secondary or tertiary biological safety cabinet. The biosafety cabinet should be placed away from doors, room ventilation shutters, and walking areas in the laboratory. It should be tested and certified at least annually to ensure that the air filtered by the HEPA filter discharged from the safety cabinet can enter the laboratory cycle. When the secondary biosafety cabinet discharges air through the building exhaust system, the connection of the exhaust duct of the safety cabinet should avoid interference with the air balance of the safety cabinet or the air balance of the building exhaust system. When using a Class III biosafety cabinet, connect directly to the exhaust system. If the three-level safety cabinet is connected to the gas supply system, the safety cabinet should be prevented from being pressurized forward.
When the operation cannot be carried out in the biosafety cabinet, personal protective equipment such as masks, masks and other protective equipment should be used. Other supporting instruments in the laboratory are also equipped with safety covers, closed equipment or other physical protection facilities. For example, a centrifuge with a safety cover or a sealed rotor, a bioreactor with a safety cover, etc., see the relevant description later in this article.
Facilities for the treatment of experimental waste are best placed in the laboratory (ie pressure cookers, chemical disinfectants, incinerators and other viable disinfection methods). Garbage should be sealed when transported out of the laboratory and should not be transported through public access.
In addition, the light in the laboratory should be suitable for all work, avoiding reflections and flashes, so as not to interfere with vision. The laboratory should also be equipped with an eye wash device.
There are some special requirements for the equipment needed to establish a P3/P4 biosafety laboratory. Some of the major and commonly used equipment are listed below:
1) Secondary/Grade 3 Biosafety Cabinet The secondary safety cabinet of the P3/P4 laboratory shall have an efflux system that requires the exhaust air to be completely and effectively filtered and discharged into the atmosphere. The MaxiSafe 2010 Class II Biosafety Cabinet and the MSC III Class III Biosafety Cabinet of Denmark's Heto-Holten A/S, represented by Gene Company, are ideal safety cabinets widely used by authoritative organizations such as the National NIH (National Health Center). Among them, the MaxiSafe 2010 Class II biosafety cabinet was certified to DIN 12950 and EN 12469 in 2000. Different from other brands, the system is equipped with three HEPA filters, two on the top of the workbench and one on the bottom. The filtration efficiency is up to 99.999%, which ensures the minimum pollution of the work area and the service life of the filter. It is the world during SARS. The necessary equipment for virus operation recommended by the World Health Organization (WHO).
2) Centrifuge
Centrifugal equipment in the P3/P4 laboratory is required to have an aerosol-proof sealing cap or to operate in a safety enclosure. In this respect, Germany's EPPENDORF's 5804/5810 series of high-speed centrifuges meet the most stringent IEC 1010-2-020 safety standards, ensuring a high degree of safety in centrifugal operations, while other centrifuges on the market generally only achieve the mandatory IEC1010-2. standard.
3) Autoclave Japan Tomy's program-controlled automatic steam disinfection cabinet is automatically controlled by the program. It has a low water level alarm device, which not only meets the laboratory standard operation requirements, but also has a cover design, stainless steel material, easy to disinfect and clean. The most important thing is to have an automatic silicon strip to seal the door when the pressure rises, which is safer.
4) Ultra-low temperature refrigerator Denmark's Heto-Holten A/S-85°C vertical ultra-low temperature refrigerator has a unique laminate cooling function to better protect samples.
French Jouan Robotics' fully automatic refrigerator Molbank (4 ° C or -20 ° C) / Biobank (-80 ° C) can be controlled by computer software to control the built-in robot to put the sample from the automatically opened small door to the designated position, or sample from the specified position In this operation, at the same time, the identity of the sample is also recognized by the bar code reader provided by the robot, thereby achieving traceability of the operation. The interior of the refrigerator is scientifically planned and can be used for storage of various samples such as microplates, deep well plates and test tube racks, and is automatically encoded by a computer to identify the position and save information. It can also be used in conjunction with the Thermo CRS robot to achieve external transfer. The instrument is connected online to achieve fully automatic operation, to avoid human error to the utmost extent.
5) Freeze-drying equipment The main equipment freeze-drying machine requires all interfaces to be well sealed. It is recommended to have all stainless steel seamless cold traps to effectively capture water vapor, easy to disinfect and durable. Denmark's Heto-Holten A/S is a well-known company with more than 40 years of experience in the production of freeze-drying equipment. They are committed to the development and production of high-quality, safe and comfortable laboratory equipment that meets the requirements of modern laboratories, especially in Temperature control, vacuum and clean technology are always at the forefront. Their vacuum concentration and freeze-drying series are carefully selected and durable. Its unique modular design, compact structure, easy operation and flexible combination can fully meet the user's various needs. Therefore, among many freeze-drying machine manufacturers, users are accustomed to flexible use and excellent cost performance, and are widely used in laboratories, pilot plants and industrial production.
6) Other conventional equipment In addition, there are some laboratory routine equipment: such as homogenizer, mixer, agitator, ultrasonic breaker, tissue grinder and micro-sampler, etc., all according to the requirements of biosafety laboratories. It is regularly disinfected and cleaned, so in addition to stable performance, leakage prevention, easy disinfection and cleaning are the main indicators. For example, the Eppendorf company's Reference micro-sampler series can be autoclaved and widely used by many biosafety laboratories around the world.
7) Special equipment for nucleic acid detection-Quantitative PCR system For the detection of pathogenic nucleic acid, quantitative PCR is a widely used technology in recent years, especially for SARS, avian influenza virus, dengue fever and other subjects are nucleic acid pathogenic sources, and the sensitivity of quantitative PCR is high. It is easy to operate and fast, and it has a good application in disease research or detection. Gene Co., Ltd. provides a complete set of equipment and related reagents from its upstream blood sample preservation and automatic viral nucleic acid purification, to fully automated PCR, reaction system preparation (automatic loading), multi-channel quantitative PCR reaction and detection. (http://)
Attachment: US Centers for Disease Control Laboratory Biosafety Level Standard: Biosafety Laboratory Classification National Institutes of Health, US Centers for Disease Management, Pathway Operation, Level 1 Barrier, Secondary Barrier
P1 BSL-1 Microbial operation that does not often trigger healthy adult disease standards does not require open lab benches, sinks
P2 BSL—2 Human pathogenic bacteria, which are dangerous due to skin wounds, inhalation, and mucous membrane exposure. BSL-1 operation plus: 1. Restricted entry 2. Biohazard warning sign 3. “Sharp device” safety measures 4. Biosafety manual, It provides waste disinfection and medical observation level 1 and 2 biological safety cabinet test clothes, gloves, and if necessary, face protection measures BSL-1 plus: autoclave
P3 BSL-3 endogenous and exogenous pathogens, which can be transmitted by aerosols, can cause serious consequences or life-threatening BSL-3 BSL-2 operation plus: 1, control entry 2, all waste disinfection 3, before washing, experiment Disinfection 4, basic serum level 1 and 2 biosafety cabinet protective laboratory clothes, gloves, if necessary, take respiratory protection measures BSL-2 plus: 1, and enter the corridor to separate 2, double door to enter, the door automatically closes 3. The discharged air does not circulate. 4. Negative pressure in the laboratory.
P4 BSL—4 A dangerous or exogenous source of high risk to life: fatal, laboratory infection by aerosol: or unknown source of risk associated with BSL3 operation: 1. Change clothes before entering 2 Laboratory front shower 3, all materials for the facility disinfection level 3 biosafety cabinet or level 1 and level 2 biosafety cabinet plus full body positive air pressure protection suit BSL3 plus: 1, separate building or isolation area 2 There are gas supply system, exhaust system, vacuum system, disinfection system 3, other relevant requirements

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