Laboratory Biosafety Levels (BL-1 to 3)

Last update January 5, 2008

The following practices and procedures apply to projects and laboratories not involving recombinant DNA molecules. The biosafety level requirements for rDNA research are listed in Appendices B3, B4, and B5.

BL-1 (Biosafety Level 1) practices, safety equipment, and facilities are appropriate for projects with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. This would apply, for example, to undergraduate and secondary educational training as well as teaching laboratories. BL-1 is appropriate for agents assigned to RG-1.

BL-2 (Biosafety Level 2) practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in experiments conducted on the open bench, provided the potential for producing splashes or aerosols is low. BL-2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. Please refer to the section on Bloodborne Pathogens for more information on OSU’s occupational program. In general, BL-2 is appropriate for agents assigned to RG-2, unless specific procedures and tasks require a higher level of containment.

BL-3 (Biosafety Level 3) practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission (e.g., Mycobacterium tuberculosis), and which may cause serious and potentially lethal infection. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols. All BL-3 work is currently limited to OSU’s Research Containment Facility.

Biosafety Level 1 (BL-1)

Standard Microbiological Practices (BL-1)

• Access to the laboratory is limited or restricted at the discretion of the Principal Investigator (PI) or laboratory director (supervisor) when experiments are in progress.
• Persons wash their hands after they handle viable materials and animals, after removing gloves and before exiting the laboratory.
• Eating, drinking, smoking, handling contact lenses and applying cosmetics are not permitted in the work area (e.g., laboratory) See OSU’s No Food No Drink Policy. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, Michigan, and federal regulations, before removal from the facility.
• An insect and rodent control program is in effect. Contact Pest Management at 7-4038 for more information.

Safety Equipment (BL-1)

• Special containment equipment is generally not required for manipulations of agents assigned to BL-1.
• It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
• Gloves should be worn if the skin on the hands is broken or if a rash exists.
• Protective eyewear should be worn for anticipated splashes of microorganisms or other hazardous materials to the face.

Laboratory Facilities (BL-1)

• Each laboratory contains a sink for hand washing.
• The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill is extremely difficult to achieve.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
• Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning.
• If the laboratory has windows that open, they are fitted with fly screens.

Biosafety Level 2 (BL-2)

Standard Microbiological Practices (BL-2)

• Access to the laboratory is limited or restricted at the discretion of the Principal Investigator (PI) or laboratory director (supervisor) when experiments are in progress.
• Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.
• Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work area (e.g., laboratory). See OSU’s No Food No Drink Policy. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, Michigan, and federal regulations, before removal from the facility.
• An insect and rodent control program is in effect. Contact Pest Management at 7-4038 for more information.

Special Practices (BL-2)

• Access to the laboratory is limited or restricted by the laboratory director or PI when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The laboratory director or PI has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
• The laboratory director or PI establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.
• All areas operating at BL-2 need to have the universal biohazard symbol (Figure 1) attached to the main entrance door(s). When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering the laboratory (Figure 2).
• Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine, PPD test).
• When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at- risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.
• OSU’s biosafety manual is adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures outlined in the biosafety manual.
• Laboratory personnel receive appropriate training through the laboratory director or PI on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. In addition, attendance at the EH&S biosafety training is recommended.
• A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative. such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.
• Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located, OSU approved, "Sharps" containers (see Biosafety Resources). Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
• Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.
• Contaminated broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps and disposed of in "Sharps" containers. Sharps filled 2/3 need to be disposed of and will be picked-up by EH&S. For more information refer to the Biohazardous Waste section.
• Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping. For more information refer to the Transportation of Biological Materials section.
• Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated and accompanied by OSU’s Equipment Release Form (Appendix H) before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, Michigan, or federal regulations, before removal from the facility.
• Spills and accidents which result in overt exposures to infectious materials are immediately reported to the laboratory director or PI. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.
• Animals not involved in the work being performed are not permitted in the lab.

Safety Equipment (Primary Barriers, BL-2)

• Properly maintained biological safety cabinets (BSC), preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:
• Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
• High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.
• All BSCs are certified annually according to National Sanitation Foundation (NSF) Standard 49, by NSF certified personnel.
• Face protection (goggles, mask, face shield or other splatter guards) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face, when the microorganisms must be manipulated outside the BSC.
• Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered; it should never be taken home by personnel.
• Gloves are worn when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed.
• Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and are not worn outside the laboratory. Disposable gloves are not washed or reused.

Laboratory Facilities (Secondary Barriers, BL-2)

• Each laboratory contains a sink for hand washing.
• The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill is extremely difficult to achieve.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
• Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
• If the laboratory has windows that open, they are fitted with fly screens.
• A method for decontamination of infectious or regulated laboratory wastes is available (e.g.. autoclave, chemical disinfection, incinerator, or other approved decontamination system).
• An eyewash facility is readily available.

Biosafety Level 3 (BL-3)

Standard Microbiological Practices (BL-3)

• Access to the laboratory or containment room is limited or restricted at the discretion of the Principal Investigator (PI), laboratory director or facility manager when experiments are in progress.
• Persons wash their hands after they handle infectious materials and animals, after removing gloves, and before leaving the laboratory.
• Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory or containment room (see OSU’s No Food No Drink Policy). Persons who wear contact lenses in these areas should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
• Mouth pipetting is prohibited; mechanical pipetting devices are used.
• All procedures are performed carefully to minimize the creation of splashes or aerosols.
• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated off-site from the laboratory are packaged in accordance with applicable local, Michigan, and federal regulations, before removal from the facility.
• An insect and rodent control program is in effect. Contact Pest Management at 7-4038 for more information.

Special Practices (BL-3)

• Laboratory doors are kept closed when experiments are in progress.
• The laboratory director, PI or facility manager controls access to the laboratory and containment rooms, restricting access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The laboratory director/PI in cooperation with the facility manager has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory or the containment room(s).
• The laboratory director/PI in cooperation with the facility manager or PI establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures, enter the laboratory or animal rooms.
• When infectious materials or infected animals are present in the laboratory or containment room, a biohazard warning sign, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures. All animal rooms require an AUCAUC approved Animal Use Form attached to the main access door(s).
• Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine, PPD test).
• Baseline serum samples for laboratory and other at-risk personnel may be collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.
• OSU’s biosafety manual is adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures as outlined in the biosafety manual.
• Laboratory personnel receive appropriate training through the laboratory director or PI on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. In addition, attendance at the EH&S biosafety training is mandatory for BL-3 work.
• The laboratory director or PI is responsible for insuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.
• A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.
• Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located, MSU approved, "Sharps" containers. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
• Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.
• Contaminated broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps and disposed of in "Sharps" containers. Sharps containers filled 2/3 need to be disposed of and will be picked-up by the ORCBS. For more information refer to the Biohazardous Waste section.
• All manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment room. No work in open vessels is conducted on the open bench.
• Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated and accompanied by OSU’s Equipment Release Form (Appendix H) before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, Michigan, or federal regulations, before removal from the facility. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.
• Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping. For more information refer to the Transportation of Biological Materials section.
• All potentially contaminated waste materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.
• Spills of infectious materials are contained, decontaminated and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material.
• Spills and accidents which result in overt or potential exposures to infectious materials are immediately reported to the laboratory director or PI and EH&S. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.
• Animals not involved in the work being performed are not permitted in the lab.

Safety Equipment (Primary Barriers, BL-3)

• Properly maintained biological safety cabinets (BSCs) are used (Class II or III) for all manipulation of infectious materials. All BSCs are certified annually according to NSF Standard 49, by NSF certified personnel.
• Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators), in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals).
• This equipment (see above) must be used for manipulations of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.
• Face protection (goggles and mask, or face shield) is worn for manipulations of infectious materials outside of a biological safety cabinet.
• Respiratory protection is worn when aerosols cannot be safely contained (i.e., outside of a biological safety cabinet), and in rooms containing infected animals. If respiratory protection is required, all personnel involved need to be included in OSU's Respiratory Protection Program.
• Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered.
• Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse.

Laboratory Facilities (Secondary Barriers, BL-3)

• The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passage way.
• Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.
• The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontamination.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
• Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
• Windows in the laboratory are closed and sealed.
• A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method).
• A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building, and is discharged to the outside with filtration and other treatment optional. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper.
• The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or Ill biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection) that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months according to NSF Standard 49.
• Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory.
• Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent, which are routinely maintained and replaced as needed.
• An eyewash facility is readily available.