The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Table 1 of this chapter and discussed in Chapter 2. The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community. Standard microbiological practices are common to all laboratories. Special microbiological practices enhance worker safety, environmental protection, and address the risk of handling agents requiring increasing levels of containment.

Laboratory Biosafety Levels

Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by appropriate risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.

The following standard practices, safety equipment, and facility requirements apply to BSL-1:

  1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
  2. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
  4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
  5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
    1. Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
    2. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
    3. Non disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
  6. Perform all procedures to minimize the creation of splashes and/or aerosols.
  7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container and secured for transport.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
  9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. The sign may include the name of the agent(s) in use, and the name and phone number of the laboratory supervisor or other responsible personnel. Agent information should be posted in accordance with the institutional policy.
  10. An effective integrated pest management program is required. See Chapter 15.
  11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child- bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
None required.
  1. Special containment devices or equipment, such as BSCs, are not generally required.
  2. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
  3. Wear protective eyewear when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials. Persons who wear contact lenses in laboratories should also wear eye protection.
  4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Wash hands prior to leaving the laboratory. In addition, BSL-1 workers should:
    1. Change gloves when contaminated, integrity has been compromised, or when otherwise necessary.
    2. Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
    3. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
  1. Laboratories should have doors for access control.
  2. Laboratories must have a sink for hand washing.
  3. The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.
  4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
    1. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
    2. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
  5. Laboratories windows that open to the exterior should be fitted with screens.
Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. It differs from BSL-1 in that 1) laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures; 2) access to the laboratory is restricted when work is being conducted; and 3) all procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment.

The following standard and special practices, safety equipment, and facility requirements apply to BSL-2:

  1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
  2. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
  4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
  5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
    1. Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
    2. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
    3. Non-disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
  6. Perform all procedures to minimize the creation of splashes and/or aerosols.
  7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container and secured for transport.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
  9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. Posted information must include: the laboratory’s biosafety level, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Agent information should be posted in accordance with the institutional policy.
  10. An effective integrated pest management program is required. See Chapter 15.
  11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
  1. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
  2. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory.
  3. When appropriate, a baseline serum sample should be stored.
  4. A laboratory-specific biosafety manual must be prepared and adopted as policy. The biosafety manual must be available and accessible.
  5. The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents.
  6. Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
  7. Laboratory equipment should be routinely decontaminated, as well as, after spills, splashes, or other potential contamination.
    1. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
    2. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
  8. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety safety manual. All such incidents must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained.
  9. Animals and plants not associated with the work being performed must not be permitted in the laboratory.
  10. All procedures involving the manipulation of infectious materials that may generate an aerosol should be conducted within a BSC or other physical containment devices.
  1. Properly maintained BSCs (preferably Class II), other appropriate personal protective equipment, or other physical containment devices must be used whenever:
    1. Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intra-nasally, and harvesting infected tissues from animals or eggs.
    2. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups.
  2. Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use must be worn while working with hazardous materials. Remove protective clothing before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). Dispose of protective clothing appropriately, or deposit it for laundering by the institution. It is recommended that laboratory clothing not be taken home.
  3. Eye and face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms must be handled outside the BSC or containment device. Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories should also wear eye protection.
  4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves must not be worn outside the laboratory. In addition, BSL-2 laboratory workers should:
    1. Change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
    2. Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
    3. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
  5. Eye, face and respiratory protection should be used in rooms containing infected animals as determined by the risk assessment.
  1. Laboratory doors should be self-closing and have locks in accordance with the institutional policies.
  2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically operated. It should be located near the exit door.
  3. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.
  4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
    1. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
    2. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
  5. Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.
  6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.
  7. Vacuum lines should be protected with High Efficiency Particulate Air (HEPA) filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
  8. An eyewash station must be readily available.
  9. There are no specific requirements on ventilation systems. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory.
  10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated back into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation must be verified.
  11. A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method).

To designate your lab as a BSL-2 space follow the procedure and checklist in Chapter 4, Section 1.

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through inhalation route exposure. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and associated procedures.

All procedures involving the manipulation of infectious materials must be conducted within BSCs, other physical containment devices, or by personnel wearing appropriate personal protective equipment.

A BSL-3 laboratory has special engineering and design features.

The following standard and special safety practices, equipment, and facility requirements apply to BSL-3:

  1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
  2. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
  4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
  5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
    1. Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
    2. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
    3. Non-disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
  6. Perform all procedures to minimize the creation of splashes and/or aerosols.
  7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method. A method for decontaminating all laboratory wastes should be available in the facility, preferably within the laboratory (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container and secured for transport.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
  9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. Posted information must include the laboratory’s biosafety level, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Agent information should be posted in accordance with the institutional policy.
  10. An effective integrated pest management program is required. See Chapter 15.
  11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
  1. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
  2. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory.
  3. Each institution should consider the need for collection and storage of serum samples from at-risk personnel.
  4. A laboratory-specific biosafety manual must be prepared and adopted as policy. The biosafety manual must be available and accessible.
  5. The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-3 agents.
  6. Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
  7. Laboratory equipment should be routinely decontaminated, as well as, after spills, splashes, or other potential contamination.
    1. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
    2. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
  8. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety safety manual. All such incidents must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained.
  9. Animals and plants not associated with the work being performed must not be permitted in the laboratory.
  10. All procedures involving the manipulation of infectious materials must be conducted within a BSC, or other physical containment devices. No work with open vessels is conducted on the bench. When a procedure cannot be performed within a BSC, a combination of personal protective equipment and other containment devices, such as a centrifuge safety cup or sealed rotor, must be used.
  1. All procedures involving the manipulation of infectious materials must be conducted within a BSC (preferably Class II or Class III), or other physical containment devices.
  2. Protective laboratory clothing with a solid-front such as tie-back or wraparound gowns, scrub suits, or coveralls are worn by workers when in the laboratory. Protective clothing is not worn outside of the laboratory. Reusable clothing is decontaminated with appropriate disinfectant before being laundered. Clothing is changed when contaminated.
  3. Eye and face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials. Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories must also wear eye protection.
  4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves must not be worn outside the laboratory. In addition, BSL-3 laboratory workers should:
    1. Change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
    2. Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
    3. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
  5. Eye, face, and respiratory protection must be used in rooms containing infected animals.
  1. Laboratory doors must be self- closing and have locks in accordance with the institutional policies.
    The laboratory must be separated from areas that are open to unrestricted traffic flow within the building.
    Access to the laboratory is restricted to entry by a series of two self-closing doors.
    A clothing change room (anteroom) may be included in the passageway between the two self- closing doors.
  2. Laboratories must have a sink for hand washing. The sink must be hands-free or automatically operated. It should be located near the exit door.
    If the laboratory is segregated into different laboratories, a sink must also be available for hand washing in each zone.
    Additional sinks may be required as determined by the risk assessment.
  3. The laboratory must be designed so that it can be easily cleaned and decontaminated. Carpets and rugs are not permitted. Seams, floors, walls, and ceiling surfaces should be sealed. Spaces around doors and ventilation openings should be capable of being sealed to facilitate space decontamination.
    1. Floors must be slip resistant, impervious to liquids, and resistant to chemicals. Consideration should be given to the installation of seamless, sealed, resilient or poured floors, with integral cove bases.
    2. Walls should be constructed to produce a sealed smooth finish that can be easily cleaned and decontaminated.
    3. Ceilings should be constructed, sealed, and finished in the same general manner as walls.

    Decontamination of the entire laboratory should be considered when there has been gross contamination of the space, significant changes in laboratory usage, for major renovations, or maintenance shut downs. Selection of the appropriate materials and methods used to decontaminate the laboratory must be based on the risk assessment of the biological agents in use.

  4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment must be accessible for cleaning.
    1. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
    2. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
  5. All windows in the laboratory must be sealed.
  6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, heavily traveled laboratory areas, and other possible airflow disruptions.
  7. Vacuum lines must be protected with HEPA filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
  8. An eyewash station must be readily available in the laboratory.
  9. A ducted air ventilation system is required. This system must provide sustained directional airflow by drawing air into the laboratory from “clean” areas toward “potentially contaminated” areas. The laboratory shall be designed such that under failure conditions the airflow will not be reversed.
    1. Laboratory personnel must be able to verify directional air flow. A visual monitoring device which confirms directional air flow must be provided at the laboratory entry. Audible alarms should be considered to notify personnel of air flow disruption.
    2. The laboratory exhaust air must not re-circulate to any other area of the building.
    3. The laboratory building exhaust air should be dispersed away from occupied areas and from building air intake locations or the exhaust air must be HEPA filtered.
  10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation must be verified. BSCs should be certified at least annually to assure correct performance. Class III BSCs must be directly (hard) connected up through the second exhaust HEPA filter of the cabinet. Supply air must be provided in such a manner that prevents positive pressurization of the cabinet.
  11. A method for decontaminating all laboratory wastes should be available in the facility, preferably within the laboratory (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method).
  12. Equipment that may produce infectious aerosols must be contained in devices that exhaust air through HEPA filtration or other equivalent technology before being discharged into the laboratory. These HEPA filters should be tested and/or replaced at least annually.
  13. Facility design consideration should be given to means of decontaminating large pieces of equipment before removal from the laboratory.
  14. Enhanced environmental and personal protection may be required by the agent summary statement, risk assessment, or applicable local, state, or federal regulations. These laboratory enhancements may include, for example, one or more of the following; an anteroom for clean storage of equipment and supplies with dress-in, shower-out capabilities; gas tight dampers to facilitate laboratory isolation; final HEPA filtration of the laboratory exhaust air; laboratory effluent decontamination; and advanced access control devices such as biometrics. HEPA filter housings should have gas-tight isolation dampers; decontamination ports; and/or bag-in/bag-out (with appropriate decontamination procedures) capability. The HEPA filter housing should allow for leak testing of each filter and assembly. The filters and the housing should be certified at least annually.
  15. The BSL-3 facility design, operational parameters, and procedures must be verified and documented prior to operation. Facilities must be re-verified and documented at least annually.
Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, aerosol transmission, or related agent with unknown risk of transmission. Agents with a close or identical antigenic relationship to agents requiring BSL-4 containment must be handled at this level until sufficient data are obtained either to confirm continued work at this level, or re-designate the level. Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Laboratory staff must understand the primary and secondary containment functions of standard and special practices, containment equipment, and laboratory design characteristics. All laboratory staff and supervisors must be competent in handling agents and procedures requiring BSL-4 containment. Access to the laboratory is controlled by the laboratory supervisor in accordance with institutional policies.

There are two models for BSL-4 laboratories:

  1. A Cabinet Laboratory where all handling of agents must be performed in a Class III BSC.
  2. A Suit Laboratory where personnel must wear a positive pressure protective suit.

BSL-4 Cabinet and Suit Laboratories have special engineering and design features to prevent microorganisms from being disseminated into the environment.

The following standard and special safety practices, equipment, and facilities apply to BSL-4:

  1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
  2. All persons leaving the laboratory must be required to take a personal body shower.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
  4. Mechanical pipetting devices must be used.
  5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Precautions, including those listed below, must be taken with any sharp items. These include:
    1. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
    2. Use of needles and syringes or other sharp instruments should be restricted in the laboratory, except when there is no practical alternative.
    3. Used needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal or decontamination. Used disposable needles must be carefully placed in puncture-resistant containers used for sharps disposal, located as close to the point of use as possible.
    4. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries.
  6. Perform all procedures to minimize the creation of splashes and/or aerosols.
  7. Decontaminate work surfaces with appropriate disinfectant after completion of work and after any spill or splash of potentially infectious material.
  8. Decontaminate all wastes before removal from the laboratory by an effective and validated method.
  9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. Posted information must include the laboratory’s biosafety level, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Agent information should be posted in accordance with the institutional policy.
  10. An effective integrated pest management program is required. See Chapter 15.
  11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
  1. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements in accordance with institutional policies.
    Only persons whose presence in the facility or individual laboratory rooms is required for scientific or support purposes should be authorized to enter.
    Entry into the facility must be limited by means of secure, locked doors. A logbook, or other means of documenting the date and time of all persons entering and leaving the laboratory must be maintained.
    While the laboratory is operational, personnel must enter and exit the laboratory through the clothing change and shower rooms except during emergencies. All personal clothing must be removed in the outer clothing change room. Laboratory clothing, including undergarments, pants, shirts, jumpsuits, shoes, and gloves, must be used by all personnel entering the laboratory. All persons leaving the laboratory must take a personal body shower. Used laboratory clothing must not be removed from the inner change room through the personal shower. These items must be treated as contaminated materials and decontaminated before laundering.
    After the laboratory has been completely decontaminated, necessary staff may enter and exit without following the clothing change and shower requirements described above.
  2. Laboratory personnel and support staff must be provided appropriate occupational medical service including medical surveillance and available immunizations for agents handled or potentially present in the laboratory. A system must be established for reporting and documenting laboratory accidents, exposures, employee absenteeism and for the medical surveillance of potential laboratory-associated illnesses. An essential adjunct to such an occupational medical services system is the availability of a facility for the isolation and medical care of personnel with potential or known laboratory acquired infections.
  3. Each institution must establish policies and procedures describing the collection and storage of serum samples from at-risk personnel.
  4. A laboratory-specific biosafety manual must be prepared. The biosafety manual must be available, accessible, and followed.
  5. The laboratory supervisor is responsible for ensuring that laboratory personnel:
    1. Demonstrate high proficiency in standard and special microbiological practices, and techniques for working with agents requiring BSL-4 containment.
    2. Receive appropriate training in the practices and operations specific to the laboratory facility.
    3. Receive annual updates or additional training when procedural or policy changes occur.
  6. Removal of biological materials that are to remain in a viable or intact state from the laboratory must be transferred to a non-breakable, sealed primary container and then enclosed in a non-breakable, sealed secondary container. These materials must be transferred through a disinfectant dunk tank, fumigation chamber, or decontamination shower. Once removed, packaged viable material must not be opened outside BSL-4 containment unless inactivated by a validated method.
  7. Laboratory equipment must be routinely decontaminated, as well as after spills, splashes, or other potential contamination.
    1. Spills involving infectious materials must be contained, decontaminated, and cleaned up by appropriate professional staff, or others properly trained and equipped to work with infectious material. A spill procedure must be developed and posted within the laboratory.
    2. Equipment must be decontaminated using an effective and validated method before repair, maintenance, or removal from the laboratory. The interior of the Class III cabinet as well as all contaminated plenums, fans and filters must be decontaminated using a validated gaseous or vapor method.
    3. Equipment or material that might be damaged by high temperatures or steam must be decontaminated using an effective and validated procedure such as a gaseous or vapor method in an airlock or chamber designed for this purpose.
  8. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety manual. All incidents must be reported to the laboratory supervisor, institutional management and appropriate laboratory personnel as defined in the laboratory biosafety manual. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained.
  9. Animals and plants not associated with the work being performed must not be permitted in the laboratory.
  10. Supplies and materials that are not brought into the BSL-4 laboratory through the change room must be brought in through a previously decontaminated double-door autoclave, fumigation chamber, or airlock. After securing the outer doors, personnel within the laboratory retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors must be secured after materials are brought into the facility. The doors of the autoclave are interlocked in a manner that prevents opening of the outer door unless the autoclave has been operated through a decontamination cycle. The doors of a fumigation chamber must be secured in a manner that prevents opening of the outer door unless the fumigation chamber has been operated through a fumigation cycle.
    Only necessary equipment and supplies should be stored inside the BSL-4 laboratory. All equipment and supplies taken inside the laboratory must be decontaminated before removal from the facility.
  11. Daily inspections of essential containment and life support systems must be completed and documented before laboratory work is initiated to ensure that the laboratory is operating according to established parameters.
  12. Practical and effective protocols for emergency situations must be established. These protocols must include plans for medical emergencies, facility malfunctions, fires, escape of animals within the laboratory, and other potential emergencies. Training in emergency response procedures must be provided to emergency response personnel and other responsible staff according to institutional policies.

Cabinet Laboratory

  1. All manipulations of infectious materials within the facility must be conducted in the Class III biological safety cabinet.
    Double-door, pass through autoclaves must be provided for decontaminating materials passing out of the Class III BSC(s). The autoclave doors must be interlocked so that only one can be opened at any time and be automatically controlled so that the outside door to the autoclave can only be opened after the decontamination cycle has been completed.
    The Class III cabinet must also have a pass-through dunk tank, fumigation chamber, or equivalent decontamination method so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the cabinet. Containment must be maintained at all times.
    The Class III cabinet must have a HEPA filter on the supply air intake and two HEPA filters in series on the exhaust outlet of the unit. There must be gas tight dampers on the supply and exhaust ducts of the cabinet to permit gas or vapor decontamination of the unit. Ports for injection of test medium must be present on all HEPA filter housings.
    The interior of the Class III cabinet must be constructed with smooth finishes that can be easily cleaned and decontaminated. All sharp edges on cabinet finishes must be eliminated to reduce the potential for cuts and tears of gloves. Equipment to be placed in the Class III cabinet should also be free of sharp edges or other surfaces that may damage or puncture the cabinet gloves.
    Class III cabinet gloves must be inspected for leaks periodically and changed if necessary. Gloves should be replaced annually during cabinet recertification. The cabinet should be designed to permit maintenance and repairs of cabinet mechanical systems (refrigeration, incubators, centrifuges, etc.) to be performed from the exterior of the cabinet whenever possible.
    Manipulation of high concentrations or large volumes of infectious agents within the Class III cabinet should be performed using physical containment devices inside the cabinet whenever practical. Such materials should be centrifuged inside the cabinet using sealed rotor heads or centrifuge safety cups.
    The Class III cabinet must be certified at least annually.
  2. Protective laboratory clothing with a solid-front such as tie-back or wraparound gowns, scrub suits, or coveralls must be worn by workers when in the laboratory. No personal clothing, jewelry, or other items except eyeglasses should be taken past the personal shower area. All protective clothing must be removed in the dirty side change room before showering. Reusable clothing must be autoclaved before being laundered.
  3. Eye, face and respiratory protection should be used in rooms containing infected animals as determined by the risk assessment. Prescription eyeglasses must be decontaminated before removal through the personal body shower.
  4. Gloves must be worn to protect against breaks or tears in the cabinet gloves. Gloves must not be worn outside the laboratory. Alternatives to latex gloves should be available. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste.

Suit Laboratory

  1. All procedures must be conducted by personnel wearing a one-piece positive pressure suit ventilated with a life support system.
    All manipulations of infectious agents must be performed within a BSC or other primary barrier system.
    Equipment that may produce aerosols must be contained in devices that exhaust air through HEPA filtration before being discharged into the laboratory. These HEPA filters should be tested annually and replaced as needed.
    HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations.
  2. Protective laboratory clothing such as scrub suits must be worn by workers before entering the room used for donning positive pressure suits. All protective clothing must be removed in the dirty side change room before entering the personal shower. Reusable laboratory clothing must be autoclaved before being laundered.
  3. Inner gloves must be worn to protect against break or tears in the outer suit gloves. Disposable gloves must not be worn outside the change area. Alternatives to latex gloves should be available. Do not wash or reuse disposable gloves. Inner gloves must be removed and discarded in the inner change room prior to personal shower. Dispose of used gloves with other contaminated waste.
  4. Decontamination of outer suit gloves is performed during operations to remove gross contamination and minimize further contamination of the laboratory.

Cabinet Laboratory

  1. The BSL-4 cabinet laboratory consists of either a separate building or a clearly demarcated and isolated zone within a building. Laboratory doors must have locks in accordance with the institutional policies.
    Rooms in the facility must be arranged to ensure sequential passage through an inner (dirty) changing area, a personal shower and an outer (clean) change room prior to exiting the room(s) containing the Class III BSC(s).
    An automatically activated emergency power source must be provided at a minimum for the laboratory exhaust system, life support systems, alarms, lighting, entry and exit controls, BSCs, and door gaskets. Monitoring and control systems for air supply, exhaust, life support, alarms, entry and exit, and security systems should be on an uninterrupted power supply (UPS).
    A double-door autoclave, dunk tank, fumigation chamber, or ventilated anteroom/airlock must be provided at the containment barrier for the passage of materials, supplies, or equipment.
  2. A hands-free sink must be provided near the door of the cabinet room(s) and the inner change room. A sink must be provided in the outer change room. All sinks in the room(s) containing the Class III BSC and the inner (dirty) change room must be connected to the wastewater decontamination system.
  3. Walls, floors, and ceilings of the laboratory must be constructed to form a sealed internal shell to facilitate fumigation and prohibit animal and insect intrusion. The internal surfaces of this shell must be resistant to liquids and chemicals used for cleaning and decontamination of the area. Floors must be monolithic, sealed and coved.
    All penetrations in the internal shell of the laboratory and inner change room must be sealed.
    Openings around doors into the cabinet room and inner change room must be minimized and capable of being sealed to facilitate decontamination.
    Drains in the laboratory floor (if present) must be connected directly to the liquid waste decontamination system.
    Services, plumbing or otherwise, that penetrate the laboratory walls, floors, ceiling, plumbing or otherwise, must ensure that no backflow from the laboratory occurs. These penetrations must be fitted with two (in series) backflow prevention devices. Consideration should be given to locating these devices outside of containment. Atmospheric venting systems must be provided with two HEPA filters in series and be sealed up to the second filter.
    Decontamination of the entire cabinet must be performed using a validated gaseous or vapor method when there have been significant changes in cabinet usage, before major renovations or maintenance shut downs, and in other situations, as determined by risk assessment. Selection of the appropriate materials and methods used for decontamination must be based on the risk assessment of the biological agents in use.
  4. Laboratory furniture must be of simple construction, capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment must be accessible for cleaning and decontamination. Chairs and other furniture should be covered with a non- porous material that can be easily decontaminated.
  5. Windows must be break-resistant and sealed.
  6. If Class II BSCs are needed in the cabinet laboratory, they must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. Class II cabinets should be located away from doors, heavily traveled laboratory areas, and other possible airflow disruptions.
  7. Central vacuum systems are not recommended. If, however, there is a central vacuum system, it must not serve areas outside the cabinet room. Two in-line HEPA filters must be placed near each use point. Filters must be installed to permit in-place decontamination and replacement.
  8. An eyewash station must be readily available in the laboratory.
  9. A dedicated non-recirculating ventilation system is provided. Only laboratories with the same HVAC requirements (i.e., other BSL-4 labs, ABSL-4, BSL-3 Ag labs) may share ventilation systems if each individual laboratory system is isolated by gas tight dampers and HEPA filters.
    The supply and exhaust components of the ventilation system must be designed to maintain the laboratory at negative pressure to surrounding areas and provide differential pressure/directional airflow between adjacent areas within the laboratory.
    Redundant supply fans are recommended. Redundant exhaust fans are required. Supply and exhaust fans must be interlocked to prevent positive pressurization of the laboratory.
    The ventilation system must be monitored and alarmed to indicate malfunction or deviation from design parameters. A visual monitoring device must be installed near the clean change room so proper differential pressures within the laboratory may be verified.
    Supply air to and exhaust air from the cabinet room, inner change room, and fumigation/decontamination chambers must pass through HEPA filter(s). The air exhaust discharge must be located away from occupied spaces and building air intakes.
    All HEPA filters should be located as near as practicable to the cabinet or laboratory in order to minimize the length of potentially contaminated ductwork. All HEPA filters must to be tested and certified annually.
    The HEPA filter housings should be designed to allow for in situ decontamination and validation of the filter prior to removal. The design of the HEPA filter housing must have gas-tight isolation dampers; decontamination ports; and ability to scan each filter assembly for leaks.
  10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment if the cabinet is tested and certified at least annually and operated according to the manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation must be verified.
    Class III BSCs must be directly and independently exhausted through two HEPA filters in series. Supply air must be provided in such a manner that prevents positive pressurization of the cabinet.
  11. Pass through dunk tanks, fumigation chambers, or equivalent decontamination methods must be provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the cabinet room(s). Access to the exit side of the pass-through shall be limited to those individuals authorized to be in the BSL-4 laboratory.
  12. Liquid effluents from cabinet room sinks, floor drains, autoclave chambers, and other sources within the cabinet room must be decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer.
    Decontamination of all liquid wastes must be documented. The decontamination process for liquid wastes must be validated physically and biologically. Biological validation must be performed annually or more often if required by institutional policy.
    Effluents from showers and toilets may be discharged to the sanitary sewer without treatment.
  13. A double-door, pass through autoclave(s) must be provided for decontaminating materials passing out of the cabinet laboratory. Autoclaves that open outside of the laboratory must be sealed to the primary wall. This bioseal must be durable and airtight. Positioning the bioseal so that the equipment can be accessed and maintained from outside the laboratory is strongly recommended. The autoclave doors must be interlocked so that only one can be opened at any time and be automatically controlled so that the outside door to the autoclave can only be opened after the decontamination cycle has been completed.
    Gas and liquid discharge from the autoclave chamber must be decontaminated. When feasible, autoclave decontamination processes should be designed so that over-pressurization cannot release unfiltered air or steam exposed to infectious material to the environment.
  14. The BSL-4 facility design parameters and operational procedures must be documented. The facility must be tested to verify that the design and operational parameters have been met prior to operation. Facilities must also be re-verified annually. Verification criteria should be modified as necessary by operational experience.
  15. Appropriate communication systems must be provided between the laboratory and the outside (e.g., voice, fax, and computer). Provisions for emergency communication and access/egress must be considered.

Suit Laboratory

  1. The BSL-4 suit laboratory consists of either a separate building or a clearly demarcated and isolated zone within a building. Laboratory doors must have locks in accordance with the institutional policies.
    Rooms in the facility must be arranged to ensure exit by sequential passage through the chemical shower, inner (dirty) change room, personal shower, and outer (clean) changing area.
    Entry into the BSL-4 laboratory must be through an airlock fitted with airtight doors. Personnel who enter this area must wear a positive pressure suit with HEPA filtered breathing air. The breathing air systems must have redundant compressors, failure alarms and emergency backup.
    A chemical shower must be provided to decontaminate the surface of the positive pressure suit before the worker leaves the laboratory. In the event of an emergency exit or failure of chemical shower system a method for decontaminating positive pressure suits, such as a gravity fed supply of chemical disinfectant, is needed.
    An automatically activated emergency power source must be provided at a minimum for the laboratory exhaust system, life support systems, alarms, lighting, entry and exit controls, BSCs, and door gaskets. Monitoring and control systems for air supply, exhaust, life support, alarms, entry and exit, and security systems should be on a UPS.
    A double-door autoclave, dunk tank, or fumigation chamber must be provided at the containment barrier for the passage of materials, supplies, or equipment.
  2. Sinks inside the suit laboratory should be placed near procedure areas and contain traps and be connected to the wastewater decontamination system.
  3. Walls, floors, and ceilings of the laboratory must be constructed to form a sealed internal shell to facilitate fumigation and prohibit animal and insect intrusion. The internal surfaces of this shell must be resistant to liquids and chemicals used for cleaning and decontamination of the area. Floors must be monolithic, sealed and coved.
    All penetrations in the internal shell of the laboratory, suit storage room and the inner change room must be sealed.
    Drains, if present in the laboratory floor, must be connected directly to the liquid waste decontamination system. Sewer vents and other service lines must be protected by two HEPA filters in series and have protection against insect and animal intrusion.
    Services, plumbing or otherwise, that penetrate the laboratory walls, floors, ceiling, plumbing or otherwise, must ensure that no backflow from the laboratory occurs. These penetrations must be fitted with two (in series) backflow prevention devices. Consideration should be given to locating these devices outside of containment. Atmospheric venting systems must be provided with two HEPA filters in series and be sealed up to the second filter.
    Decontamination of the entire laboratory must be performed using a validated gaseous or vapor method when there have been significant changes in laboratory usage, before major renovations or maintenance shut downs, and in other situations, as determined by risk assessment.
  4. Laboratory furniture must be of simple construction, capable of supporting anticipated loading and uses. Sharp edges and corners should be avoided. Spaces between benches, cabinets, and equipment must be accessible for cleaning and decontamination. Chairs and other furniture should be covered with a non-porous material that can be easily decontaminated.
  5. Windows must be break-resistant and sealed.
  6. BSCs and other primary containment barrier systems must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, heavily traveled laboratory areas, and other possible airflow disruptions.
  7. Central vacuum systems are not recommended. If, however, there is a central vacuum system, it must not serve areas outside the BSL-4 laboratory. Two inline HEPA filters must be placed near each use point. Filters must be installed to permit in-place decontamination and replacement.
  8. An eyewash station must be readily available in the laboratory area for use during maintenance and repair activities.
  9. A dedicated non-recirculating ventilation system is provided. Only laboratories with the same HVAC requirements (i.e., other BSL-4 labs, ABSL-4, BSL-3 Ag labs) may share ventilation systems if each individual laboratory system is isolated by gas tight dampers and HEPA filters.
    The supply and exhaust components of the ventilation system must be designed to maintain the laboratory at negative pressure to surrounding areas and provide differential pressure/directional airflow between adjacent areas within the laboratory.
    Redundant supply fans are recommended. Redundant exhaust fans are required. Supply and exhaust fans must be interlocked to prevent positive pressurization of the laboratory.
    The ventilation system must be monitored and alarmed to indicate malfunction or deviation from design parameters. A visual monitoring device must be installed near the clean change room so proper differential pressures within the laboratory may be verified.
    Supply air to the laboratory, including the decontamination shower, must pass through a HEPA filter. All exhaust air from the suit laboratory, decontamination shower and fumigation or decontamination chambers must pass through two HEPA filters, in series, before discharge to the outside. The exhaust air discharge must be located away from occupied spaces and air intakes.
    All HEPA filters must be located as near as practicable to the laboratory in order to minimize the length of potentially contaminated ductwork. All HEPA filters must be tested and certified annually.
    The HEPA filter housings should be designed to allow for in situ decontamination and validation of the filter prior to removal. The design of the HEPA filter housing must have gas-tight isolation dampers; decontamination ports; and ability to scan each filter assembly for leaks.
  10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated back into the laboratory environment if the cabinet is tested and certified at least annually and operated according to the manufacturer’s recommendations. Biological safety cabinets can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation must be verified.
  11. Pass through dunk tanks, fumigation chambers, or equivalent decontamination methods must be provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the BSL-4 laboratory. Access to the exit side of the pass-through shall be limited to those individuals authorized to be in the BSL-4 laboratory.
  12. Liquid effluents from chemical showers, sinks, floor drains, autoclave chambers, and other sources within the laboratory must be decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer.
    Decontamination of all liquid wastes must be documented. The decontamination process for liquid wastes must be validated physically and biologically. Biological validation must be performed annually or more often if required by institutional policy.
    Effluents from personal body showers and toilets may be discharged to the sanitary sewer without treatment.
  13. A double-door, pass through autoclave(s) must be provided for decontaminating materials passing out of the cabinet laboratory. Autoclaves that open outside of the laboratory must be sealed to the primary wall. This bioseal must be durable and airtight. Positioning the bioseal so that the equipment can be accessed and maintained from outside the laboratory is strongly recommended. The autoclave doors must be interlocked so that only one can be opened at any time and be automatically controlled so that the outside door to the autoclave can only be opened after the decontamination cycle has been completed.
    Gas and liquid discharge from the autoclave chamber must be decontaminated. When feasible, autoclave decontamination processes should be designed so that over-pressurization cannot release unfiltered air or steam exposed to infectious material to the environment.
  14. The BSL-4 facility design parameters and operational procedures must be documented. The facility must be tested to verify that the design and operational parameters have been met prior to operation. Facilities must also be re-verified annually. Verification criteria should be modified as necessary by operational experience.
  15. Appropriate communication systems must be provided between the laboratory and the outside (e.g., voice, fax, and computer). Provisions for emergency communication and access/egress should be considered.

Table 1: Summary of Recommended Biosafety Levels for Infectious Agents

Download the Summary of Recommended Biosafety Levels for Infectious Agents.

Agents Practices Primary Barriers and
Safety Equipment
Facilities
(Secondary Barriers)
BSL-1 Not known to consistently cause diseases in healthy adults Standard MicrobiologicalPractices PPEs*

  • Laboratory coats; gloves; closed-toed shoes; face protection as needed
Open bench and sink required
BSL-2
  • Agents associated with human disease
  • Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure
BSL practice plus:

  • Limited access
  • Biohazard warning signs
  • “Sharps”precautions
  • Biosafety manual defining any needed waste decontamination or medical surveillance policies
Primary barriers:

  • Class I or II BSCs or other physical containmentdevices used for all manipulations of agents that cause splashes or aerosols of infectious materials

PPEs*:

  • Laboratory coats; gloves; closed toed shoes; face protection as needed
BSL-1 plus:

  • Autoclave available
BSL-3
  • Indigenous or exotic agents with potential for aerosol transmission
  • Disease may have serious or lethalconsequences
BSL‐2 practice plus:

  • Controlled access
  • Decontamination of all waste
  • Decontamination of laboratory clothing before laundering
  • Baseline serum
Primary barriers:

  • Class I or II BSCs or other physical containment devices used for all open manipulation of agents

PPEs*:

  • Protective laboratory clothing; gloves; respiratory protection as needed
BSL-2 plus:

  • Physical separation from access corridors
  • Self‐closing, double‐door access
  • Exhaust air not recirculated
  • Negative airflow into laboratory
BSL-4 UNC does not have BSL – 4 Facilities

*PPE – Personal Protective Equipment

Section I: Designating Biological Safety Level 2 at UNC

The first step in designating BSL-2 space at UNC is notifying EHS of your desire to do so. This is done by submitting an up-to-date copy of your Laboratory Safety Plan (LSP).

Access all current LSP documents.

The Lab Safety Plan can be daunting; to complete the requirements for each section properly, refer to the general UNC Laboratory Safety Manual, Chapter 2. The latest copy of the Lab Safety Manual is available.

For BSL-2 consideration, the Schedule F (Biological Hazards) of the L ab Safety Plan is completed and submitted to EHS for approval. The Lab Safety Plan is to be updated annually with EHS.

Recombinant DNA research on campus probably falls into one of the three following groups that may require registration with the UNC Institutional Biosafety Committee (IBC):

  • Exempt Experiments
  • Experiments Requiring Prior Approval
  • Experiments Requiring IBC Notice Simultaneous with Initiation

Examples of each are available online. Access forms and other information may be accessed.

All biological hazards listed on the Schedule F will be reviewed annually with all laboratory workers including those handling and/or treating biohazard waste. The annual in-house training form (or an equivalent) will be used to document this training and kept in the lab safety binder. These requirements are reviewed with new staff and the Schedule F is reviewed annually. Accessing this website allows lab workers to view all lab members’ training requirements.
All workers exposed to human source materials will complete Bloodborne Pathogens (BBP) training annually. OSHA defines workers as exposed to BBP if they work with human blood or other potentially infectious material (including human cell lines-continuous or primary.)

Complete the online self-study BBP training. You may want to forward this link along to any lab workers who will require this training.

All workers to which the OSHA Bloodborne Pathogen standard applies should have, or officially decline, the Hepatitis B vaccination series at the University Employee Occupational Health Clinic.

For more information about the risk of human tissue and the Bloodborne Pathogen Standard please refer to the EHS website.

For more information about the Hepatitis B vaccination series, view the CDC fact sheet.

Laboratory doors are self-closing and have locks in accordance with the institutional policies. The Principal Investigator is ultimately responsible for the control of, and access to, laboratories where risk group 2 agents are stored or manipulated (guidelines).
BSL-2 entryway signs are posted for lab spaces in which large volumes or high concentrations of risk group 2 agents are present. Also, posting entryway signs for all other BSL-2 lab space is required (e.g. for areas where human blood or other potentially infectious material including human cell lines is present).
The lab space designated at BSL-2 has a sink available for hand washing. The sink may be manual, hands-free, or automatically operated and should have soap and disposable paper towels readily available at all times for washing hands at the sink. Ideally, the sink is located near the exit door.
The laboratory should be designed so that it can be easily cleaned and decontaminated. This can be difficult in older buildings that were designed without present day biosafety precautions in mind. Carpets and rugs in laboratories are not permitted. Check areas for worn and damaged bench tops or flooring that may harbor microbes in the event of a spill.

To have Facilities Services repair the area, speak with your department’s business manager and/or submit an online service request.

Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning. To have Facilities Services repair an area, speak with your department’s business manager and/or submit an online service request.
Chairs used at the biological safety cabinet must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant. Cloth covered chairs should not be used at the biosafety cabinet. The chairs must be capable of supporting anticipated loads and uses. This practice should be applied to chairs at lab benches too.
Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens. To have Facilities Services fit or repair openings to the exterior of the building, speak with your department’s business manager and/or submit an online service request.
EHS must be consulted regarding the placement and use of your biological safety cabinet. Contact us to arrange an appointment. Biological safety cabinets (BSC), (aka “tissue culture hoods”) must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions. To have Facilities Services install deflectors at room air vents, speak with your department’s business manager and/or submit an online service request. Technically, if a BSC has passed certification in place with the vents running, deflectors are not necessary however, EHS recommends not leaving it to chance…remember, that air barrier is pretty delicate on BSCs and seasonal airflow variations in laboratory buildings can be significant.
Vacuum lines located at biosafety cabinets (aka: “tissue culture hoods”) must have protection via an absorbent or liquid disinfectant trap and a High Efficiency Particulate Air (HEPA) filter, or its equivalent to prevent contamination of the vacuum system. Filters must be replaced as needed. This practice should also apply to aspirating liquid at the BSL-2 benchtop. Order info is available at www.fishersci.com; search for part # 09-744-75 or # 09-744-76.
To be compliant with BSL-2 standards at UNC, eyewash stations must be plumbed units that meet the ANSI Standard Z358.1-2004 such as: Personal eye flush squeeze bottles do not meet ANSI requirements, because they cannot deliver the required minimum flow rate and duration. EHS discourages the presence of these bottles particularly in BSL-2 labs because they have a limited shelf life, are prone to contamination, and are ineffective at dual-eye or eye-face irrigation. Facilities Services can install a deck-mounted unit at an existing sink (place work orders online). EHS recommends the following unit to compensate for the distance and obstructions to the nearest safety shower from some labs: Fisher Scientific deck mount eye wash #S47711. For more information about eyewash facilities in UNC labs, see Chapter 3 of the UNC Laboratory Safety Manual.
A shower facility, other than emergency drench hoses, must be located in the building. To compensate for the distance and obstructions to the nearest safety shower from some designated BSL-2 space, EHS recommends the following unit: Fisher Scientific deck mount eye wash #S47711.
There are no specific requirements on ventilation systems in BSL-2 labs at UNC. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory. Chapter 7-4 (Facility Requirements) of the general UNC Laboratory Safety Manual indicates the following:

“Exhaust ventilation systems are designed to maintain an inflow of air from the corridor into the work area. The exhaust air from the work area must discharge directly to the outdoors, and clear of occupied buildings and air intakes. Exhaust air from the work area must not recirculate. The exhaust air from glove boxes must filter through high-efficiency particulate air (HEPA) and charcoal filters. EHS shall determine the need for and type of treatment for other primary containment equipment. Exhaust air treatment systems that remove toxic chemicals from the exhaust air by collection mechanism such as filtration or absorption must operate in a manner that permits maintenance, to avoid direct contact with the collection medium. All exhaust air from primary containment equipment must discharge directly to the outdoors and disperse clear of occupied buildings and intakes.”

Because most biological safety cabinets (BSC) at UNC re-circulate HEPA-filtered exhaust air into the laboratory environment, the cabinet should be tested and certified at least annually and operated according to manufacturer’s recommendations. Never use hazardous chemicals in these cabinets, the vapors bypass the HEPA (particulate) filters and enter your breathing zone.

Some BSCs on campus may be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation should be verified annually. Only minute amounts of hazardous chemicals should be used in an exhausted cabinet. More information about biological safety cabinets is available in the UNC Biological Safety Manual.

In UNC laboratories, an autoclave must be accessible to decontaminate all biohazard waste before disposal. This varies from the UNC Hospitals requirement for biohazard waste because UNC Hospitals incinerates all biohazard waste. If an autoclave is not accessible to your lab, contact EHS.

Refer to the EHS Biohazard Waste Management website for waste decontamination requirements.

Refer to the “Liquids” section of the Biohazard Waste Disposal Chart. If your liquid waste was used for propagating microbes/viral vectors/toxins AND you are unable to autoclave your liquid biohazard waste, you will need to make application to the North Carolina Medical Waste Division to dispose of this chemically disinfected liquid microbiological waste down the drain. For more information, refer to the EHS Chemical Treatment of Liquid Microbiological Waste website.

The Request for Approval to Chemical Treat Liquid Microbiological Waste form is available.

Laboratory equipment used for BSL-2 containment is posted with the universal biohazard warning symbol (to communicate hazard to maintenance workers, visitors, etc.). This symbol is used to identify the actual or potential presence of a biological hazard on or in freezers, incubators, centrifuges, biological safety cabinets, etc. which are used with agents listed on Schedule F of the Laboratory Safety Plan. Biohazard warning labels may be printed on a color printer from the EHS website or they may be ordered from www.fishersci.com, search for reorder # 18-999-934.
Because most biological safety cabinets (BSC) (aka “tissue culture hoods”) on campus re- circulate HEPA filtered exhaust air into the laboratory environment, the cabinet should be tested and certified at least annually and operated according to manufacturer’s recommendations. Never use hazardous chemicals in these cabinets, the vapors bypass the HEPA filter to enter your breathing zone. Some BSCs on campus can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation should be verified annually. Only minute amounts of hazardous chemicals should be used in an exhausted cabinet.

To arrange for certification of your biological safety cabinet or to inquire about UNC covering the cost, contact EHS.

Biological safety cabinets (BSC) (aka “tissue culture hoods”) must be used whenever procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intra-nasally, and harvesting infected tissues from animals or eggs.

BSCs must be used whenever procedures with high concentrations or large volumes of infectious agents are conducted. Such materials should only be centrifuged in the open laboratory when sealed rotor heads or centrifuge safety cups are used.

Many activities associated with centrifuges may create significant amounts of infectious aerosol, including: filling centrifuge tubes; removing plugs or caps from tubes after centrifugation; removing supernatant; re-suspending sedimented pellets; breakage of tubes during centrifugation; and centrifugation itself. Follow these steps to prevent the generation of aerosols in centrifuges:

  1. Routinely inspect the centrifuge to ensure there is no leakage.
  2. Do not overfill centrifuge tubes.
  3. Wipe the outside of the tubes with an appropriate disinfectant after they are filled and sealed.
  4. Centrifuge inside a biological safety cabinet. If a biological safety cabinet is not available, internal aerosol containment devices (e.g., sealed canisters, safety cups or buckets with covers, heat sealed tubes or sealed rotors) should be used.
  5. Remove aerosol containment devices and open them in a biological safety cabinet. If the biological safety cabinet is in use, a minimum of 10 minutes settling time should be allowed before opening.
Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use are worn while working in designated BSL-2 space. Protective clothing is removed before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). Protective clothing is disposed of appropriately, or deposited for laundering by the institution (more information). Laboratory clothing may not be taken home.

Chapter 5-7 of the general UNC Laboratory Safety Manual has more information regarding laboratory clothing, protective apparel and laundering and UNC.

Eye and face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms must be handled outside the BSC or containment device. Eye and face protection is disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories also wear eye protection.

Chapter 4 of the campus safety manual details UNC’s laboratory eye and face protection policy.

Special care is taken to avoid skin contamination at BSL-2. Gloves are worn to protect hands when handling experimental animals and when skin contact with the agent is unavoidable. Glove selection is based on an appropriate risk assessment. Alternatives to latex gloves are available. Gloves are not worn outside the laboratory. The general UNC Laboratory Safety Manual (Chapter 5-5) details requirements for the use of gloves in laboratories. In addition, BSL-2 laboratory workers should:

  1. Change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
  2. Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
  3. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
Eye, face and respiratory protection is used in rooms containing infected animals as determined by the risk assessment and EHS.

See the general UNC Laboratory Safety Manual Chapter 14 for safe handling of laboratory animals.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from lab animals and diaphragm bottles. Only needle-locking syringes or disposable syringe- needle units (i.e., needle is integral to the syringe) are used at BSL-2. Extreme caution is used when handling needles and syringes to avoid auto inoculation and the generation of aerosols during use and disposal. Needles are not to be bent, sheared, replaced in the needle sheath or guard, or removed from the syringe following use.

Needles and syringes are promptly placed into appropriately labeled plastic sharps containers. Red containers are clearly marked as “Biohazardous Sharps” and autoclaved (remember to mark an “X” with autoclave tape directly over the biohazard warning label) prior to disposal or, if the sharps are not biohazardous, white or clear plastic containers are labeled “Nonhazardous Sharps”.

When ordering the plastic sharps containers online from www.fishersci.com, please use catalog # 1482664B and/or 14830124.

Labels for these containers can be obtained from the EHS Safety Labels web page.

For other biohazardous waste collection and disposal methods, a Disposal Chart is available at the EHS website.

Fisher Scientific is currently contracted by UNC to supply storerooms on campus. A Fisher representative can also help you find a product to meet your needs.

Contact info: Jeremy Crosson, Sales Representative
Fisher Scientific
919-673-6801
919-843-5604 UNC customer service
919-843-5605 fax
919-962-2160 Fisher chemistry storeroom
jeremy.crosson@thermofisher.com
http://www.fishersci.com/

All persons entering the designated BSL-2 area are advised of the potential hazards and they meet specific entry/exit requirements when manipulations involving materials from the Lab Safety Plan’s Schedule F (biological hazards) are taking place. These requirements are reviewed with new staff and the Schedule F is reviewed annually as part of the lab’s annual in-house training.

Before exiting the BSL-2 laboratory for non-laboratory areas, lab coats, gowns, smocks, and gloves are removed and left in the laboratory. All workers wash their hands after they de-glove and prior to exiting the lab after they handle materials involving viable material.

Each laboratory must establish policies and procedures describing the collection and storage of serum samples from at-risk personnel as appropriate. If applicable, this will be listed on the Lab Safety Plan’s Schedule F (biological hazards) under “Medical Surveillance.” For more information, contact EHS biological safety or the University Employee Occupational Health Clinic. Procedures for animal surveillance are discussed in Chapter 14-9 of the general UNC Laboratory Safety Manual.
Laboratory personnel are provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. If applicable, this should be listed on the Lab Safety Plan’s Schedule F (biological hazards) under “Medical Surveillance.”

If your group is working with a known pathogen for which there is an effective vaccine, the vaccine should be made available to all workers. For more information, contact EHS biological safety or the University Employee Occupational Health Clinic.

Anyone in the lab working with (including treating waste) human blood or other potentially infectious material (including human cell lines, established, or primary) is required by the OSHA Bloodborne Pathogen standard to have, seek, or officially decline the Hepatitis B vaccination series. For questions regarding this and other vaccination series, please contact the University Employee Occupational Health Clinic. CDC Hepatitis B vaccination information is available at the CDC website.

For more information about the risk of human tissue and the Bloodborne Pathogen Standard please refer to the EHS website.

Procedures for animal bites and immunizations are discussed in Chapter 14-6 of the general UNC Laboratory Safety Manual.

The UNC Biosafety Manual and Exposure Control Plan has been adopted as laboratory policy and the Schedule F (Biological hazards) is reviewed with each worker at least annually during annual in-house training. A paper copy of the Biosafety Manual and Schedule F must be available and accessible to workers at all times and documentation of the annual in-house training is available in the lab safety binder.

The latest copy of the UNC Laboratory Exposure Control Plan is available online (for now, print the General Version) and keep in your safety binder.

The Principal Investigator has the final responsibility for determining who may enter or work in the BSL-2 space and for advising persons of the potential hazard and entry requirements (e.g. immunization) for entry. The Principal Investigator has ensured that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents. At a minimum, this includes training in aseptic techniques and in the biology of the organisms used in the experiment so that the potential biohazards can be understood and appreciated. All procedures are performed carefully to minimize the creation of aerosols.

Eating, drinking, smoking and applying cosmetics are not permitted in the work area. Food will be stored and consumed in designated areas (labels) used for this purpose only. These requirements are reviewed with new staff and the Schedule F is reviewed annually as part of the lab’s annual in-house training.

For more information about animal handling technical proficiency, see the general UNC Laboratory Safety Manual, Chapter 14-3.

Potentially infectious materials will be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility. A leak-proof box, preferably equipped with a gasket seal lid, is used for transport of potentially infectious materials from one location to another on campus. This is particularly important when moving samples from patient care areas in UNC Hospitals to the lab. Containers such as igloo coolers or Rubbermaid containers will suffice provided they have enough absorbent material placed inside and a biohazard warning label on the outermost container.
Laboratory equipment and work surfaces are decontaminated once a day and after any spill of viable material. The appropriate disinfectant and recommended contact time is listed on the Lab Safety Plan’s Schedule F (biological hazards) under “Safety Precautions.”

The lab has determined how and where the decontamination of all cultures, stocks, and other potentially infectious materials will be performed before disposal. This is listed on your Lab Safety Plan’s Schedule F (biological hazards) under “Safety Precautions.” Refer to the EHS Biohazard Waste Management website for waste decontamination requirements.

Prior to repair, maintenance, or removal from the laboratory, equipment will be decontaminated.

Workers are properly trained and equipped to contain, decontaminate, and clean up spills involving infectious material. The emergency plan that describes the procedures to be followed if an accident contaminates personnel or the environment is listed on the Lab Safety Plan’s Schedule F (biological hazards) under #8 “Emergency Procedures.” At a minimum, bleach is provided in the lab space and plenty of paper towels are available for spill clean-up purposes.

Refer to the EHS Biohazard Waste Management website for waste decontamination requirements.

Incidents that may result in overt exposures to materials handled at BSL-2 will be immediately evaluated and treated according to procedures described in this manual. All such incidents must be reported to the laboratory supervisor, the University Employee Occupational Health Clinic and the Institutional Biosafety Committee.
Animals and plants not associated with the work being performed are not permitted in the laboratory. This is also consistent with the Campus Safety Manual, Chapter 2.
All procedures involving the manipulation of infectious materials that may generate an aerosol will be conducted within a biological safety cabinet (aka “tissue culture hood”) or other physical containment device.

Appendix A: Biological Safety Level 2 Checklist

Use the Biosafety Level 2 checklist for guidance when designating new BSL‐2 lab space at UNC-Chapel Hill. This checklist is designed to answer common/general questions during the set‐up process. Registration of your lab space is required by EHS and can be achieved by completing an online Laboratory Safety Plan. Feel free to consult the online Biological Safety Manual or contact EHS for guidance.

Download the Laboratory checklist for designating Biological Safety Level 2 at UNC.

Appendix B: Cryogenic Preservation of Biological Materials

Liquid nitrogen dewars are commonly utilized for cryogenic preservation of biological materials.

However, liquid nitrogen exhibits a boiling point of -195.8° C and expands over 600 fold when brought to room temperature. Cryogenic preservation vials or “cryovials” stored in the liquid phase of liquid nitrogen can rupture upon warming if liquid nitrogen has infiltrated them, resulting in an explosion hazard. Pieces of the cryovial may be propelled towards personnel resulting in physical injury, particularly to the eyes, and exposure to the cryovial’s contents. For this reason, cryogenic storage of hazardous materials including infectious agents and biological toxins is strongly discouraged. Contact the Biological Safety Officer for guidance.

Safety Practices

Exposure to liquid nitrogen may damage living tissue. Personnel must be properly attired including the use of long pants or skirts and closed toed shoes. Additional required Personal Protective Equipment (PPE) includes lab coats, disposable gloves, thermal outer gloves and eye/face protection such as safety glasses and/or a face shield.

Utilize screw capped cryogenic preservation vials with rubber O rings designed specifically for cryogenic preservation. Ensure caps are tightly screwed on. Store cryovials in the gaseous phase above the liquid nitrogen to avoid infiltration. Ensure laboratory personnel are aware of the explosion hazard to avoid overfilling liquid nitrogen dewars to the point of submerging cryovials.

When removing cryovials from the dewar, immediately place them in a sealed and leak-proof container to contain a possible explosion. Example secondary containers include 50 mL conical tubes or Tupperware containers. Rubber ice buckets do not constitute an appropriate secondary container. If desired, ice or dry ice can be placed in the secondary container or the secondary container can be placed in a larger container with ice or dry ice. The explosion hazard will no longer exist moments after bringing the cryovial to room temperature.

When replenishing liquid nitrogen dewars, work in a properly ventilated area to avoid an asphyxiation hazard. Patiently watch over the dewar to avoid overflow. Spilled liquid nitrogen can damage living tissue as well as facilities and equipment.

Emergency Exposures/Spills

If a cryogenic liquid is spilled and leads to skin or eye exposure immediate medical treatment is required. Contact the University Employee Occupational Health Clinic (919-966-9119) during work hours for immediate medical care or call 911. Large spills of cryogenic liquids can lead to an oxygen-deficient environment. Personnel should immediately evacuate the area and call 911.

Back to Chapter Three
Proceed to Chapter Five