Biosafety Level 2

Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents that represent a moderate hazard for personnel and the environment. It differs in that

a.

laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists,

b.

access to the laboratory is limited when work is being conducted, and

c.

certain procedures in which infectious aerosols are created are conducted in biological safety cabinets or other physical containment equipment.

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2:

A. Standard microbiological practices

1.

Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress.

2.

Work surfaces are decontaminated at least once a day and after any spill of viable material.

3.

All infectious liquid or solid waste is decontaminated before being disposed of.

4.

Mechanical pipetting devices are used; mouth pipetting is prohibited.

5.

Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food must be stored in cabinets or refrigerators designed and used for this purpose only. Food storage cabinets or refrigerators should be located outside the work area.

6.

Persons are to wash their hands when they leave the laboratory after handling infectious material or animals.

7.

All procedures are performed carefully to minimize the creation of aerosols.

B. Special practices

1.

Contaminated materials that are to be decontaminated away from the laboratory are placed in a durable, leakproof container that is closed before being removed from the laboratory.

2.

The laboratory director limits access to the laboratory. 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. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

3.

The laboratory director establishes policies or procedures whereby only persons who have been advised of the potential hazard and who meet any specific entry requirements (e.g., vaccination) enter the laboratory or animal rooms.

4.

When an infectious agent being worked with in the laboratory requires special provisions for entry (e.g., vaccination), a hazard warning sign that incorporates 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.

5.

An insect and rodent control program is in effect.

6.

Laboratory coats, gowns, smocks, or uniforms are worn while in the laboratory. Before leaving the laboratory for nonlaboratory areas (e.g., cafeteria, library, administrative offices), this protective clothing is removed and left in the laboratory or covered with a clean coat not used in the laboratory.

7.

Animals not involved in the work being performed are not permitted in the laboratory.

8.

Special care is taken to avoid having skin be contaminated with infectious material; gloves should be worn when handling infected animals and when skin contact with infectious material is unavoidable.

9.

All waste from laboratories and animal rooms is appropriately decontaminated before disposal.

10.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluid. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.

11.

Spills and accidents that result in overt exposures to infectious material are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained.

12.

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 on the function of the facility.

13.

A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read instructions on practices and procedures and to follow them.

C. Containment equipment

Biological safety cabinets (Class I or II) or other appropriate personal-protection or physical-containment devices are used when:

1.

Procedures with a high potential for creating infectious aerosols 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.

2.

High concentrations or large volumes of infectious agents are used. Some types of materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if the containers are opened only in a biological safety cabinet.

D. Laboratory facilities

1.

The laboratory is designed so that it can be easily cleaned.

2.

Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

3.

Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

4.

Each laboratory contains a sink for handwashing.

5.

If the laboratory has windows that open, they are fitted with fly screens.

6.

An autoclave for decontaminating infectious laboratory wastes is available.

Biosafety Level 3

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by inhalation. Laboratory personnel have specific training in handling pathogenic and/or potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment devices or by personnel wearing appropriate personal-protection clothing and devices. The laboratory has special engineering and design features. It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations, and directional airflow). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories in which facility features satisfy Biosafety Level 2 recommendations if the recommended "Standard Microbiological Practices," "Special Practices," and "Containment Equipment" for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director.

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 3:

A. Standard microbiological practices

1.

Work surfaces are decontaminated at least once a day and after any spill of viable material.

2.

All infectious liquid or solid waste is decontaminated before being disposed of.

3.

Mechanical pipetting devices are used; mouth pipetting is prohibited.

4.

Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area.

5.

Persons wash their hands after handling infectious materials and animals and every time they leave the laboratory.

6.

All procedures are performed carefully to minimize the creation of aerosols.

B. Special practices

1.

Laboratory doors are kept closed when experiments are in progress.

2.

Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable, leakproof container that is closed before being removed from the laboratory.

3.

The laboratory director controls access to the laboratory and limits access only to persons whose presence is required for program or support purposes. 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 director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

4.

The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., vaccination), and who comply with all entry and exit procedures enter the laboratory or animal rooms.

5.

When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign (incorporating the universal biohazard symbol) 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 vaccinations, respirators, or other personal-protection measures.

6.

All activities involving infectious materials are conducted in biological safety cabinets or other physical-containment devices within the containment module. No work is conducted in open vessels on the open bench.

7.

The work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious materials is finished. Plastic-backed paper toweling used on nonperforated work surfaces within biological safety cabinets facilitates clean-up.

8.

An insect and rodent control program is in effect.

9.

Laboratory clothing that protects street clothing (e.g., solid-front or wrap-around gowns, scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the laboratory, and it is decontaminated before being laundered.

10.

Special care is taken to avoid skin contamination with infectious materials; gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable.

11.

Molded surgical masks or respirators are worn in rooms containing infected animals.

12.

Animals and plants not related to the work being conducted are not permitted in the laboratory.

13.

All waste from laboratories and animal rooms is appropriately decontaminated before being disposed of.

14.

Vacuum lines are protected with high-efficiency particulate air (HEPA) filters and liquid disinfectant traps.

15.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Extreme caution is used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before being discarded or reused.

16.

Spills and accidents that result in overt or potential exposures to infectious material are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided, and written records are maintained.

17.

Baseline serum samples for all 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 laboratory.

18.

A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read instructions on practices and procedures and to follow them.

C. Containment equipment

Biological safety cabinets (Class I, II, or III) or other appropriate combinations of personal-protection or physical-containment devices (e.g., special protective clothing, masks, gloves, respirators, centrifuge safety cups, sealed centrifuge rotors, and containment caging for animals) are used for all activities with infectious materials that pose a threat of aerosol exposure. These include: manipulation of cultures and of clinical or environmental material that 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.

D. Laboratory facilities

1.

The laboratory is separated from areas that are open to unrestricted traffic flow within the building. Passage through two sets of doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. Physical separation of the high-containment laboratory from access corridors or other laboratories or activities may also be provided by a double-doored clothes-change room (showers may be included), airlock, or other access facility that requires passing through two sets of doors before entering the laboratory.

2.

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 decontaminating the area.

3.

Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

4.

Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

5.

Each laboratory contains a sink for washing hands. The sink is foot-, elbow-, or automatically operated and is located near the laboratory exit door.

6.

Windows in the laboratory are closed and sealed.

7.

Access doors to the laboratory or containment module are self-closing.

8.

An autoclave for decontaminating laboratory wastes is available, preferably within the laboratory.

9.

A ducted exhaust-air ventilation system is provided. This system creates directional airflow that draws air into the laboratory through the entry area. The exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow is proper (i.e., into the laboratory). The exhaust air from the laboratory room can be discharged to the outside without being filtered or otherwise treated.

10.

The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets is discharged directly to the outside or through the building exhaust system. Exhaust air from Class I or II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. If the HEPA-filtered exhaust air from Class I or II biological safety cabinets is to be discharged to the outside through the building exhaust 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.

Animal Biosafety Level 2

A. Standard practices

1.

Doors to animal rooms open inward, are self-closing, and are kept closed when infected animals are present.

2.

Work surfaces are decontaminated after use or spills of viable materials.

3.

Eating, drinking, smoking, and storing of food for human use are not permitted in animal rooms.

4.

Personnel wash their hands after handling cultures and animals and before leaving the animal room.

5.

All procedures are carefully performed to minimize the creation of aerosols.

6.

An insect and rodent control program is in effect.

B. Special practices

1.

Cages are decontaminated, preferably by autoclaving, before being cleaned and washed.

2.

Surgical-type masks are worn by all personnel entering animal rooms housing nonhuman primates.

3.

Laboratory coats, gowns, or uniforms are worn while in the animal room. This protective clothing is removed before leaving the animal facility.

4.

The laboratory or animal-facility director limits access to the animal room only to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress. In general, persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal room.

5.

The laboratory or animal-facility director establishes policies and procedures whereby only persons who have been advised of the potential hazard and who meet any specific requirements (e.g., vaccination) may enter the animal room.

6.

When an infectious agent in use in the animal room requires special-entry provisions (e.g., vaccination), a hazard warning sign (incorporating the universal biohazard symbol) is posted on the access door to the animal room. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the animal-facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

7.

Special care is taken to avoid contaminating skin with infectious material; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable.

8.

All waste from the animal room is appropriately decontaminated—preferably by autoclaving— before being disposed of. Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.

9.

Hypodermic needles and syringes are used only for the parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before being discarded or reused.

10.

If floor drains are provided, the drain taps are always filled with water or a suitable disinfectant.

11.

When appropriate, considering the agents handled, baseline serum samples from animal-care and other at-risk personnel are collected and stored. Additional serum samples may be collected periodically, depending on the agents handled or the function of the facility.

C. Containment equipment

Biological safety cabinets, other physical-containment devices, and/or personal-protection devices (e.g., respirators, face shields) are used when procedures with a high potential for creating aerosols are conducted. These include necropsy of infected animals, harvesting of infected tissues or fluids from animals or eggs, intranasal inoculation of animals, and manipulation of high concentrations or large volumes of infectious materials.

D. Animal facilities

1.

The animal facility is designed and constructed to facilitate cleaning and housekeeping.

2.

A sink for washing hands is available in the room that houses infected animals.

3.

If the animal facility has windows that open, they are fitted with fly screens.

4.

It is recommended, but not required, that the direction of airflow in the animal facility is inward and that exhaust air is discharged to the outside without being recirculated to other rooms.

5.

An autoclave that can be used for decontaminating infectious laboratory waste is available in the same building that contains the animal facility.

Animal Biosafety Level 3

A. Standard practices

1.

Doors to animal rooms open inward, are self-closing, and are kept closed when work with infected animals is in progress.

2.

Work surfaces are decontaminated after use or after spills of viable materials.

3.

Eating, drinking, smoking, and storing of food for human use are not permitted in the animal room.

4.

Personnel wash their hands after handling cultures or animals and before leaving the laboratory.

5.

All procedures are carefully performed to minimize the creation of aerosols.

6.

An insect and rodent control program is in effect.

B. Special practices

1.

Cages are autoclaved before bedding is removed and before they are cleaned and washed.

2.

Surgical-type masks or other respiratory protection devices (e.g., respirators) are worn by personnel entering rooms that house animals infected with agents assigned to Biosafety Level 3.

3.

Wrap-around or solid-front gowns or uniforms are worn by personnel entering the animal room. Front-button laboratory coats are unsuitable. Protective gowns must remain in the animal room and must be decontaminated before being laundered.

4.

The laboratory director or other responsible person limits access to the animal room only to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when infected animals are present. In general, persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal room.

5.

The laboratory director or other responsible person establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., vaccination) may enter the animal room.

6.

Hazard warning signs (incorporating the universal biohazard warning symbol) are posted on access doors to animal rooms containing animals infected with agents assigned to Biosafety Level 3 are present. The hazard warning sign should identify the agent(s) in use, list the name and telephone number of the animal room supervisor or other responsible person(s), and indicate any special conditions of entry into the animal room (e.g., the need for vaccinations or respirators).

7.

Personnel wear gloves when handling infected animals. Gloves are removed aseptically and autoclaved with other animal room waste before being disposed of or reused.

8.

All wastes from the animal room are autoclaved before being disposed of. All animal carcasses are incinerated. Dead animals are transported from the animal room to the incinerator in leakproof, covered containers.

9.

Hypodermic needles and syringes are used only for gavage or parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before being discarded or reused. When possible, cannulas should be used instead of sharp needles (e.g., for gavage).

10.

If floor drains are provided, the drain traps are always filled with water or a suitable disinfectant.

11.

If vacuum lines are provided, they are protected with HEPA filters and liquid disinfectant traps.

12.

Boots, shoe covers, or other protective footwear and disinfectant footbaths are available and used when indicated.

C. Containment equipment

1.

Personal-protection clothing and equipment and/or other physical-containment devices are used for all procedures and manipulations of infectious materials or infected animals.

2.

The risk of infectious aerosols from infected animals or their bedding can be reduced if animals are housed in partial-containment caging systems, such as open cages placed in ventilated enclosures (e.g., laminar-flow cabinets), solid-wall and -bottom cages covered by filter bonnets, or other equivalent primary containment systems.

D. Animal facilities

1.

The animal facility is designed and constructed to facilitate cleaning and housekeeping and is separated from areas that are open to unrestricted personnel traffic within the building. Passage through two sets of doors is the basic requirement for entry into the animal room from access corridors or other contiguous areas. Physical separation of the animal room from access corridors or from other activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility that requires passage through two sets of doors before entering the animal room.

2.

The interior surfaces of walls, floors, and ceilings are water resistant so that they can be cleaned easily. Penetrations in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination.

3.

A foot-, elbow-, or automatically operated sink for handwashing is provided near each animal-room exit door.

4.

Windows in the animal room are closed and sealed.

5.

Animal room doors are self-closing and are kept closed when infected animals are present.

6.

An autoclave for decontaminating wastes is available, preferably within the animal room. Materials to be autoclaved outside the animal room are transported in a covered, leakproof container.

7.

An exhaust-air ventilation system is provided. This system creates directional airflow that draws air into the animal room through the entry area. The building exhaust can be used for this purpose if the exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow is proper (i.e., into the animal room). The exhaust air from the animal room that does not pass through biological safety cabinets or other primary containment equipment can be discharged to the outside without being filtered or otherwise treated.

8.

The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets or other primary containment devices is discharged directly to the outside or through the building's exhaust system. Exhaust air from these primary containment devices may be recirculated within the animal room if the cabinet is tested and certified at least every 12 months. If the HEPA-filtered exhaust air from Class I or Class II biological safety cabinets is discharged to the outside through the building exhaust 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.