Biosafety Levels: Classification, Practices, Primary Barriers and Secondary Barriers
When working with biological contaminants, protecting just the worker is oftentimes not enough. Systems must also be in place to protect the environment and the facility from possible contamination. Depending on the type of infectious biological microorganism or laboratory animal, specific containment and safety procedures must be followed.
There are four biosafety levels which increase in safety and security measures as the dangers associated with the biologicals increase. Standard practices, special practices, primary barriers and secondary barriers are carried forward to higher biosafety levels, but may be more stringent and more precautions may be added.
Level 1 classification: Biosafety Level 1 labs are working with well-characterized agents that are not known for causing disease in healthy adult humans and are of minimal potential hazard to laboratory personal and the environment.
Level 1 standard practices: Access to the lab may be restricted, at the lab director's discretion, during experiments with cultures and specimens. Hands must be washed after handling specimens and before leaving the lab. Eating, drinking, smoking, handling contact lenses, applying cosmetics and food storage for humans is not permitted in work areas. Mouth pipetting is prohibited, mechanical pipetting devices must be used. Sharps safe-handling policies must be instituted. Work surfaces must be decontaminated once a day or after any spill of viable material. All cultures, stocks and other regulated wastes must be decontaminated before disposal by an approved decontamination method such as autoclaving.
Level 1 primary barriers/safety equipment: Biological cabinets are generally not used with this level of agent. Employees are encouraged to wear lab coats, gowns or uniforms to prevent contamination of street clothes. Gloves should be worn if skin is broken or a rash is present. Eyewear should be worn for procedures where splash of microorganisms or hazardous materials is anticipated.
Level 1 secondary barriers/laboratory facilities: Labs should have doors to limit access. Each lab needs a sink for handwashing. Design of the lab should facilitate easy cleaning, without rugs or carpets. Benchtops need to be impervious to water and be heat- and chemical-resistant.
Level 2 classification: Biosafety Level 2 labs are working with agents of moderate potential hazard to personnel and the environment. Laboratory personnel have specific training in handling pathogenic agents.
Level 2 standard practices: These remain the same as practices in Biosafety Level 1 labs with the exception of decontamination procedures of work surfaces that must be done at the completion of work, at the end of the day and after any spill or splash of viable material with disinfectants that are effective against the agents of concern.
Level 2 special practices: Access to the lab is limited or restricted by the lab director when work with infectious agents is in progress. People who are at increased risk of acquiring infection or for whom infection might have serious consequences are not allowed in the lab or animal rooms. The lab director establishes policies and procedures whereby only persons who have been advised of hazards and meet entry requirements, such as immunizations, may enter the lab. A biohazard sign must be posted on the entrance to the lab when etiologic agents are in use. Lab personnel receive appropriate immunizations or tests for the agents handled or potentially present in the lab. The lab director ensures the lab and support personnel receive appropriate training on the hazards associated with the work involved. A high degree of caution must be taken with any contaminated sharp items, including needles, syringes, slides, pipettes, capillary tubes and scalpels. Spills and accidents that result in overt exposures are immediately reported to the lab director; medical evaluation, surveillance and treatment are provided and written records are maintained.
Level 2 primary barriers/safety equipment: Biological safety cabinets, preferably Class II, should be used when potential for infectious splash or aerosols exist or high concentrations/large volumes of infectious agents are in use. Face protection, including goggles, masks, face shields or splatter guards, should be used for anticipated splashes or sprays for work that must be performed outside the biological safety cabinet. Protective clothing is worn while in the lab and removed and left in the lab before leaving for non-lab areas. All protective clothing is disposed or laundered by the institution and should never go home with personnel. Gloves are worn when hands may contact infectious materials; double-gloving may be appropriate depending on the application procedure. Disposable gloves may not be reused or washed. Hands are washed upon removal of gloves.
Level 2 secondary barriers/laboratory facilities: Facilities that house restricted agents must have lockable doors. Biological safety cabinets must be installed to ensure operation within all applicable parameters. An eye wash station should be readily available. Illumination should be adequate for all activities; avoiding reflections and glare that could impede vision.
Level 3 classification: Laboratories that fall under this category include clinical, diagnostic, teaching, research or production facilities working with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by inhalation.
Level 3 standard practices: Standard practices for Biosafety Level 3 laboratories are the same as Biosafety Level 2 laboratories.
Level 3 special practices: Lab doors are kept closed when experiments are in progress. A biosafety manual specific to the lab is prepared or adopted by the lab director. The lab director is responsible for ensuring that all personnel demonstrate proficiency in standard microbiological practices and techniques and in all practices and operations specific to the facility. All open manipulations involving infectious materials are conducted in biological safety cabinets or other containment devices. No work in open vessels is conducted on benchtops. Spills must be decontaminated, contained and cleaned by appropriately trained staff following previously developed and posted spill procedures.
Level 3 primary barriers/safety equipment: All manipulations of infectious materials are conducted in Class II or Class III biological safety cabinets. When a procedure or process cannot be conducted within a biological safety cabinet, appropriate combinations of personal protective equipment, such as respirators and eye/face protection, are used. Respiratory and face protection are used when in rooms containing infected animals.
Level 3 secondary barriers/laboratory facilities: Passage through a series of two self-closing doors is the basic requirement for entry into the lab from access corridors. Each lab contains a hands-free sink for handwashing located near the room exit door. Walls, floors and ceilings must be constructed for easy cleaning and decontamination. All windows must be closed and sealed. A ducted exhaust air ventilation system is provided. HEPA-filtered exhaust air from a Class II biological safety cabinet can be recirculated into the lab if the cabinet is tested and certified annually. Continuous flow centrifuges or other aerosol-producing equipment are contained in devices that exhaust air through HEPA filters before discharge into the lab. Vacuum lines are protected by liquid disinfectant traps and HEPA filters.
Level 4 classification: This level of protection is required when working with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted lab infections and life-threatening disease. Agents with a close or identical antigenic relationship to known Biosafety Level 4 agents are handled at this level until sufficient data are obtained to confirm work at Biosafety Level 4 or to reclassify them to a lower level.
Level 4 standard practices: Standard practices for Biosafety Level 4 laboratories are the same as Biosafety Level 2 and 3 laboratories.
Level 4 special practices: Only people whose presence in the facility or lab rooms is required for program or support purposes are authorized to enter. Personnel enter and leave the lab only through the clothing change and shower rooms. They take a decontaminating shower each time they leave the lab. Personal clothing is removed in the outer clothing changing room and kept there. Complete laboratory clothing is provided and used by all personnel entering the lab. When leaving the lab, personnel remove their lab clothing which is autoclaved before laundering. Supplies and materials needed in the facility are brought in through a double-doored autoclave, fumigation chamber or airlock. A system must be established for reporting lab accidents, exposure and employee absenteeism, and for the medical surveillance of potential lab-associated illness. Materials not related to the experiment being conducted are not permitted in the facility.
Level 4 primary barriers/safety equipment: All procedures within the facility are conducted in a Class III biological safety cabinet or in a Class II biological safety cabinet used in conjunction with one-piece positive pressure personnel suits ventilated by a life-support system.
Level 4 secondary barriers/laboratory facilities: There are two models for Biosafety Level 4 labs. A lab can follow requirements of either model or a combination of the two.
- Model 1: Cabinet Laboratory. In this model the facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms are arranged to ensure passage through a minimum of two doors prior to entering the room containing the Class III biological safety cabinet. Walls, floors and ceilings of the cabinet room and inner change room are constructed to form a sealed internal shell to allow fumigation and prevent entry of animals and insects. If there is a central vacuum system it cannot serve areas outside of the cabinet room. Access doors are self-closing and lockable. Windows are break-resistant and sealed. Plumbing and ventilation systems must decontaminate liquid and air before being allowed to leave the system. The facility design and operational procedures must be documented and the facility must be tested at least annually for verification that the design and operational parameters are met. Appropriate communication systems between the lab and outside must be provided.
- Model 2: Suit Laboratory. In this model, the facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms in the facility are arranged to ensure passage through the changing and decontamination areas prior to entering the room where work is done with the Biosafety Level 4 agents (suit area). Personnel who enter this area wear a one-piece positive pressure suit ventilated by a life-support system protected by HEPA filtration. Daily inspections of all containment parameters and life-support systems are completed before lab work is initiated. Walls, floors and ceilings of the suit room and inner change room are constructed to form a sealed internal shell to allow fumigation and prevent entry of animals and insects. If there is a central vacuum system, it cannot serve areas outside of the suit room. Access doors are self-closing and lockable. Windows are break-resistant and sealed. Plumbing and ventilation systems must decontaminate liquid and air before it is allowed to leave the system. The positioning of the supply and exhaust points should minimize dead air space in the room. Treated exhaust air from Class II biological safety cabinets, located in a facility where workers wear a positive pressure suit, may be discharged into the room environment or to the outside through facility air exhaust system. The facility design and operational procedures must be documented and the facility must be tested at least annually for verification that the design and operational parameters are met. Appropriate communication systems between the lab and outside must be provided.
For other documents related to this topic, see:
Quick Tips #254: What is the CDC?
Quick Tips #206: Disinfectants and Antiseptics
Centers for Disease Control and Prevention (CDC) - Laboratory Biosafety Level Criteria.
OSHA Lab Standard, 29 CFR 1910.1450.
Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition.
Find even more information you can use to help make informed decisions about the regulatory issues you face in your workplace every day. View all Quick Tips Technical Resources at www.grainger.com/quicktips.
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The content in this newsletter is intended for general information purposes only. This publication is not a substitute for review of the applicable government regulations and standards, and should not be construed as legal advice or opinion. Readers with specific compliance questions should refer to the cited regulation or consult with an attorney.
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