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 which may cause
serious or potentially lethal disease as a result of exposure by the
inhalation route. Laboratory personnel have specific training in
handling pathogenic and potentially lethal agents, and are supervised
by competent scientists who are experienced in working with these
All procedures involving the manipulation of infectious materials are
conducted within biological safety cabinets or other physical
containment devices, or by personnel wearing appropriate personal
protective clothing and equipment. The laboratory has special
engineering and design features.
It is recognized, however, that some existing facilities may not have
all the facility features recommended for Biosafety Level 3 (i.e.,
double-door access zone and sealed penetrations). In this
circumstance, an acceptable level of safety for the conduct of routine
procedures, (e.g., diagnostic procedures involving the propagation of
an agent for identification, typing, susceptibility testing, etc.), may
be achieved in a Biosafety Level 2 facility, providing 1) the exhaust
air from the laboratory room is discharged to the outdoors, 2) the
ventilation to the laboratory is balanced to provide directional
airflow into the room, 3) access to the laboratory is restricted when
work is in progress, and 4) the recommended Standard Microbiological
Practices, Special Practices, and Safety 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 safety practices, equipment and
facilities apply to agents assigned to Biosafety Level 3:
A. Standard Microbiological Practices
B. Special Practices
- Access to the laboratory is limited or
restricted at the discretion of the laboratory director when
experiments are in progress.
- Persons wash their hands after they handle
viable materials, after removing gloves, and before leaving the
- Eating, drinking, smoking, handling contact
lenses, and applying cosmetics are not permitted in the laboratory.
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.
- Policies for the safe handling of sharps are
- All procedures are performed carefully to
minimize the creation of splashes or aerosols
- Work surfaces are decontaminated on completion
of work or at the end of the day and after any spill or splash of
viable material with disinfectants that are effective against the
agents of concern.
- 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 placed in a
durable, leak proof container and closed for transport from the
laboratory. Infectious waste from BSL-3 laboratories should be
decontaminated before removal for off-site disposal.
- An insect and rodent control program is in
BMBL 4th edition:Appendix G).
C. Safety Equipment (Primary
- Laboratory doors are kept closed
when experiments are in progress.
- The laboratory director controls
access to the laboratory and restricts access 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 have
serious consequences are not allowed in the laboratory or animal rooms.
For example, persons who are immuno-compromised or
immuno-suppressed may be at risk of acquiring infections. The
director has the final responsibility for assessing each circumstance
and determining who may enter or work in the laboratory. No minors
should be allowed in the laboratory. (see
Minors in Laboratories policies and forms)
- 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., 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 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 immunizations, respirators, or other personal protective
- Laboratory personnel receive the
appropriate immunizations or tests for the agents handled or
potentially present in the laboratory (e.g., hepatitis B vaccine or TB
skin testing), and periodic testing as recommended for the agent being
- Baseline serum samples are
collected as appropriate and stored for all laboratory and other
at-risk personnel. Additional serum specimens may be periodically
collected, depending on the agents handled or the function of the
- A biosafety manual specific to
the laboratory is prepared or adopted by the laboratory director and
biosafety precautions are incorporated into standard operating
procedures. Personnel are advised of special hazards and are required
to read and follow instructions on practices and procedures.
- Laboratory and support personnel
receive appropriate training 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 changes.
- The laboratory director is
responsible for ensuring 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
- 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. Plastic ware should be substituted for glassware
- 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 puncture-resistant containers used for sharps disposal.
Non-disposable sharps must be placed in a hard-walled container for
transport to a processing area for decontamination, preferably by
- Syringes which re-sheathe
the needle, needleless systems, and other safe devices are used when
- 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. Containers of contaminated
needles, sharp equipment, and broken glass should be decontaminated
before disposal, and disposed of according to any local, state, or
- All open manipulations involving
infectious materials are conducted in biological safety cabinets or
other physical containment devices within the containment module. No
work in open vessels is conducted on the open bench. Clean-up is
facilitated by using plastic-backed paper toweling on non-perforated
work surfaces within biological safety cabinets.
- . Laboratory equipment and work
surfaces should be decontaminated routinely with an effective
disinfectant, after work with infectious materials is finished, and
especially after overt spills, splashes, or other contamination with
- Spills of infectious materials are
decontaminated, contained and cleaned up by appropriate professional
staff, or others properly trained and equipped to work with
concentrated infectious material. Spill procedures are developed
- Contaminated equipment must be
decontaminated before removal from the facility for repair or
maintenance or packaging for transport, in accordance with applicable
local, state, or federal regulations.
- Cultures, tissues, specimens of
body fluids, or wastes are placed in a container that prevents leakage
during collection, handling, processing, storage, transport, or
- All potentially contaminated
waste materials (e.g., gloves, lab coats, etc.) from laboratories are
decontaminated before disposal or reuse.
- Spills and accidents that result
in overt or potential exposures to infectious materials are immediately
reported to the laboratory director. Appropriate medical evaluation,
surveillance, and treatment are provided and written records are
- Animals and plants not related
to the work being conducted are not permitted in the laboratory.
D. Laboratory Facilities
- Protective laboratory clothing
such as solid-front or wrap-around gowns, scrub suits, or coveralls are
worn by workers when in the laboratory. Protective clothing is not worn
outside the laboratory. Reusable clothing is decontaminated
before being laundered. Clothing is changed when overtly
- Gloves must be worn when
handling infectious materials, infected animals, and when handling
- Frequent changing of gloves
accompanied by hand washing is recommended. Disposable gloves are not
- All manipulations of infectious
materials, necropsy of infected animals, harvesting of tissues or
fluids from infected animals or embryonate eggs , etc., are conducted
in a Class II or Class III biological safety cabinet (see Types of BSC's)
- When a procedure or process
cannot be conducted within a biological safety cabinet, then
appropriate combinations of personal protective equipment (e.g.,
respirators, face shields) and physical containment devices (e.g.,
centrifuge safety cups or sealed rotors) are used.
- Respiratory and face protection
are used when in rooms containing infected animals.
- The laboratory is separated
from areas that are open to unrestricted traffic flow within the
building, and access to the laboratory is restricted. Passage through a
series of two self-closing doors is the basic requirement for entry
into the laboratory from access corridors. Doors are lockable (see Laboratory Security). A clothes
change room may be included in the passageway.
- Each laboratory room contains a
sink for hand washing. The sink is hands-free or automatically
operated and is located near the room exit door.
- The interior surfaces of walls,
floors, and ceilings of areas where BSL-3 agents are handled are
constructed for easy cleaning and decontamination. Seams, if present,
must be sealed. Walls, ceilings, and floors should be smooth,
impermeable to liquids and resistant to the chemicals and disinfectants
normally used in the laboratory. Floors should be monolithic and
slip-resistant. Consideration should be given to the use of
coved floor coverings. Penetrations in floors, walls, and
ceiling surfaces are sealed. Openings such as around ducts and
the spaces between doors and frames are capable of being sealed to
- Bench tops are impervious to
water and are resistant to moderate heat and the organic solvents,
acids, alkalies, and those chemicals used to decontaminate the work
surfaces and equipment.
- Laboratory furniture is capable
of supporting anticipated loading and uses. Spaces between benches,
cabinets, and equipment are accessible for cleaning. Chairs and
other furniture used in laboratory work should be covered with a
non-fabric material that can be easily decontaminated.
- All windows in the laboratory
are closed and sealed.
- A method for decontaminating all
laboratory wastes is available in the facility and utilized, preferably
within the laboratory (i.e., autoclave, chemical disinfection,
incineration, or other approved decontamination method). Consideration
should be given to means of decontaminating equipment. If waste
is transported out of the laboratory, it should be properly sealed and
not transported in public corridors.
- Biological safety cabinets are
required and are located away from doors, from room supply louvers, and
from heavily-traveled laboratory areas.
- A ducted exhaust air ventilation
system is provided. This system creates directional airflow
which draws air into the laboratory from "clean" areas and toward
"contaminated" areas. The exhaust air is not recirculated to any
other area of the building. Filtration and other treatments of
the exhaust air are not required, but may be considered based on site
requirements, and specific agent manipulations and use
conditions. The outside exhaust must be dispersed away from
occupied areas and air intakes, or the exhaust must be HEPA-filtered.
Laboratory personnel must verify that the direction of the
airflow (into the laboratory) is proper. It is recommended that
a visual monitoring device that indicates and confirms directional
inward airflow be provided at the laboratory entry. Consideration
should be given to installing an HVAC control system to prevent
sustained positive pressurization of the laboratory. Audible
alarms should be considered to notify personnel of HVAC system failure.
- HEPA-filtered exhaust air from a
Class II biological safety cabinet can be recirculated into the
laboratory if the cabinet is tested and certified at least annually.
When exhaust air from Class II safety cabinets is to be discharged to
the outside through the building exhaust air system, the cabinets must
be connected in a manner that avoids any interference with the air
balance of the cabinets or the building exhaust system (e.g., an air
gap between the cabinet exhaust and the exhaust duct). When Class
III biological safety cabinets are used they should be directly
connected to the exhaust system. If the Class III cabinets are
connected to the supply system, it is done in a manner that prevents
positive pressurization of the cabinets .
- 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.
These HEPA systems are tested at least annually. Alternatively, the
exhaust from such equipment may be vented to the outside if it is
dispersed away from occupied areas and air intakes.
- Vacuum lines are protected with
liquid disinfectant traps and HEPA filters, or their equivalent.
Filters must be replaced as needed. An alternative is to use portable
vacuum pumps (also properly protected with traps and filters).
- An eyewash station is readily
available inside the laboratory.
- Illumination is adequate for all
activities, avoiding reflections and glare that could impede vision.
- The Biosafety Level 3 facility
design and operational procedures must be documented. The facility must
be tested for verification that the design and operational parameters
have been met prior to operation. Facilities should be re-verified, at
least annually, against these procedures as modified by operational
- Additional environmental
protection (e.g., personnel showers, HEPA filtration of exhaust air,
containment of other piped services and the provision of effluent
decontamination) should be considered if recommended by the agent
summary statement, as determined by risk assessment, the site
conditions, or other applicable federal, state, or local regulations.
Reproduced from "Biosafety in Microbiological and Biomedical
Laboratories, BMBL 4th Edition" with permission from the Center for
Disease Control (CDC).