Lawrence Berkeley National Laboratory masthead A-Z Index Berkeley Lab masthead U.S. Department of Energy logo Phone Book Jobs Search

Environment, Health, & Safety Division

Appendix C

Laboratory Biosafety Level 1 and 2 Criteria

 

C.1    Introduction and Scope

This appendix describes criteria for laboratory Biosafety Level 1 (BL1) and BL2 in the same manner and level of detail presented in Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition. Requirements from the NIH Guidelines and Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens (BBP) Standard were also added by LBNL to each BMBL criteria statement as needed to integrate requirements from all of these standards. LBNL requirements were also succinctly added when needed to clarify important requirements or implementation policy specifically related to BMBL criteria statements.

See Section 4.0 of this manual for additional information on biosafety principles and levels, and Section 4.4.1 for additional information on laboratory biosafety levels. See Section 5.0 of this manual for additional information and requirements on controls described in specific criteria statements.

Text Box:  C.2    Laboratory Biosafety Level 1

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. BL1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open benchtops 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 BL1:

C.2.1 BL1 Standard Microbiological Practices

  1. The laboratory supervisor and work lead must enforce LBNL institutional policies that control access to the site and laboratory facilities as described in the LBNL Site Security Plan. Policies and practices include, for example, the hosting of visitors and the issuance of gate passes, badges, and/or keys to control access to the site, building, and/or room based on each individual’s business need and experiments in progress. In addition, laboratory areas should have doors for access control.
  2. Persons must wash their hands: (a) after working with potentially hazardous materials, recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption are not 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 the 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 sharps must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
    3. Nondisposable 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. Plasticware should be substituted for glassware whenever possible.
  6. Perform all procedures to minimize splashes and/or aerosols.
  7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious or viable recombinant material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious or recombinant 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. An effective integrated pest management program is required.
  10. 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 or ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of childbearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals who have these conditions should be encouraged to identify themselves to the institution’s health care provider for appropriate counseling and guidance.

C.2.2   BL1 Special Practices

None required.

C.2.3   BL1 Safety Equipment (Primary Barriers and Personal Protective Equipment)

  1. Special containment devices or equipment, such as biosafety cabinets (BSCs), are not generally required.
  2. Protective laboratory clothing (e.g., coats, gowns, or uniforms) should be worn to prevent contamination of personal clothing.
  3. Eye protection must be worn in the laboratory and when conducting procedures that have the potential to create splashes of biological materials or other hazardous materials.
  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, BL1 workers should:
    1. Change gloves when contaminated, when their 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.

C.2.4   BL1 Laboratory Facilities (Secondary Barriers)

  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. Benchtops 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 nonporous material that can be easily cleaned and decontaminated with appropriate disinfectant.
  5. Laboratory windows that open to the exterior should be fitted with screens.
    Text Box:  Text Box:

C.3    Laboratory Biosafety Level 2

Biosafety Level 2 builds upon BL1. BL2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. It differs from BL1 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 BL2:

C.3.1 BL2 Standard Microbiological Practices

  1. The laboratory supervisor and work lead must enforce the institutional policies that control access to the site and laboratory facilities as described in the LBNL Site Security Plan. Policies and practices include, for example, the hosting of visitors and the issuance of gate passes, badges, and/or keys to control access to the site, building, and/or room based on each individual’s business need and experiments in progress. Access to the laboratory should be controlled when the laboratory is unoccupied during nonbusiness hours, (e.g., by locking doors to the laboratory areas and/or doors to the building entrance).
  2. Persons must wash their hands (a) after working with potentially hazardous materials, recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the laboratory.
  3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption are not 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, the laboratory supervisor and work lead should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Use of sharps with Risk Group (RG) 2 materials should be restricted and included in the Biological Use Authorization (BUA) as part of the risk assessment.

    Precautions, including those listed below, must always be taken with sharp items:

    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 sharps must be carefully placed in conveniently located, properly labeled, leakproof, puncture-resistant, and closable containers used for sharps disposal. Contaminated disposable sharps are disposed of immediately after use in containers that are not overfilled. These containers are closed immediately when full.
    3. Nondisposable sharps must be placed in a properly labeled, leakproof, puncture-resistant, hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. In addition, these sharps must not be stored or processed in a manner that requires workers to reach by hand into the containers where these sharps have been placed.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plasticware 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 or viable recombinant material with appropriate disinfectant.
  8. Decontaminate all cultures, stocks, and other potentially infectious or recombinant 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. When infectious agents (i.e., human pathogens) are present or there are organisms that require special provisions for entry (e.g., vaccination), additional biological hazard warning signage is required at the laboratory entrance. This signage must incorporate the universal biohazard symbol and include the laboratory’s biosafety level; the identity of the agent(s) or the words Infectious Agent(s); the name and telephone number of the supervisor, work lead, PI, or other responsible personnel; and any special requirements or procedures for entering and exiting the laboratory. The Chemical Safety Hygiene Plan (CHSP) Caution Placard will be used to accomplish these additional signage requirements. Any requirements for posting identities of agents or posting special entry and exit procedures will be specified in the BUA.
  10. An effective integrated pest management program is required.
  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, or ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of childbearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals who have these conditions should be encouraged to identify themselves to the institution’s health care provider for appropriate counseling and guidance.

C.3.2   BL2 Special Practices

  1. All persons entering the laboratory must be advised of the potential hazards and meet any specific entry/exit requirements as communicated through laboratory door postings or other means. Minimum biosafety hazard advisories include a required biohazard symbol posted at the entrance to the BL2 laboratory. Any additional biosafety requirements necessary for advising and protecting personnel entering and exiting the area will be specified in the BUA based on a risk assessment.
  2. Laboratory personnel must be provided with 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, and 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 BL2 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 decontaminated on a routine basis and after spills, splashes, or other potential contamination.
    1. Spills involving infectious materials must be contained, decontaminated, and cleaned 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. Appropriate records should be 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 device.

C.3.3   BL2 Safety Equipment (Primary Barriers and Personal Protective Equipment)

  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 intranasally, and harvesting infected tissues from animals or eggs.
    2. High concentrations or large volumes of infectious agents or organisms containing recombinant DNA are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups. In this case, the rotor heads and centrifuge cups must be opened inside a BSC.
  2. Protective laboratory clothing (e.g., coats, gowns, smocks, or uniforms) designated for laboratory use should be worn to prevent contamination of personal clothing and must be worn when working at BL2 or when working with RG2 or other hazardous materials. Remove protective clothing before leaving for nonlaboratory areas (e.g., cafeteria, library, administrative offices). Dispose of protective clothing appropriately, or deposit it for laundering services provided by an LBNL subcontractor. Laboratory clothing must not be taken home.
  3. Eye protection must be worn in the laboratory. Eye and face protection (goggles, mask, face shield, or other splatter guard) must be used when it is anticipated that splashes, sprays, splatters, or droplets of infectious or other hazardous materials may be generated and could contaminate the eyes, nose, or mouth (e.g., when RG2 microorganisms must be handled outside the BSC or containment device). This eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse.
  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 that were used in BL1 and BL2 work must not be worn outside the laboratory. In addition, BL2 laboratory workers should:
    1. Change gloves when contaminated, when their 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.

C.3.4   BL2 Laboratory Facilities (Secondary Barriers)

  1. Laboratory areas should have doors for access and ventilation control, and the doors should be self-closing and have locks designed in accordance with LBNL standards.
  2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically operated. The sink 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. Benchtops 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 nonporous 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 recirculated 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 ensure 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).