Biosafety Level 2
Biosafety Level 2 is
similar to Biosafety Level 1 and is suitable for work involving agents
of moderate potential hazard to personnel and the environment. It
differs from BSL-1 in that (1) laboratory personnel have specific
training in handling pathogenic agents and are directed by competent
scientists; (2) access to the laboratory is limited when work is being
conducted; (3) extreme precautions are taken with contaminated sharp
items; and (4) certain procedures in which infectious aerosols or
splashes may be 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
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 work areas.
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. Materials to be decontaminated off-site from the
facility are packaged in accordance with applicable local, state, and
federal regulations, before removal from the facility.
- An insect and rodent control program is in
effect (see Appendix G).
C. Safety Equipment (Primary
- Access to the laboratory is
limited or restricted by the laboratory director when work with
infectious agents is in progress. In general, 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 increased risk of acquiring infections. The
laboratory director has the final responsibility for assessing each
circumstance and determining who may enter or work in the laboratory or
- The laboratory director
establishes policies and procedures whereby only persons who have been
advised of the potential hazards and meet specific entry requirements
(e.g., immunization) may enter the laboratory.
- A biohazard sign must be posted
on the entrance to the laboratory when etiologic agents are in use.
Appropriate information to be posted includes the agent(s) in use, the
biosafety level, the required immunizations, the investigator's name
and telephone number, any personal protective equipment that must be
worn in the laboratory, and any procedures required for exiting the
- Laboratory personnel receive
appropriate immunizations or tests for the agents handled or
potentially present in the laboratory (e.g., hepatitis B vaccine or TB
- 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.
- Biosafety procedures are
incorporated into standard operating procedures or in a biosafety
manual adopted or prepared specifically for the laboratory by the
laboratory director. Personnel are advised of special hazards and are
required to read and follow instructions on practices and procedures.
- The laboratory director ensures
that 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 or policy changes.
- 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 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
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 autoclaving.
- Syringes which re-sheathe the needle,
needleless systems, and other safety devices are used when appropriate.
- 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 are decontaminated before
disposal, according to any local, state, or federal regulations.
- Cultures, tissues, specimens of
body fluids, or potentially infectious wastes are placed in a container
with a cover that prevents leakage during collection, handling,
processing, storage, transport, or shipping.
- Laboratory equipment and work
surfaces should be decontaminated with an effective 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
according to any local, state, or federal regulations before it is sent
for repair or maintenance or packaged for transport in accordance with
applicable local, state, or federal regulations, before removal from
- Spills and accidents that result
in overt exposures to infectious materials are immediately reported to
the laboratory director. Medical evaluation, surveillance, and
treatment are provided as appropriate and written records are
- Animals not involved in the work
being performed are not permitted in the lab.
D. Laboratory Facilities
- Properly maintained biological
safety cabinets, 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
intra nasally, and harvesting infected tissues from animals or
- 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.
- Face protection (goggles, mask,
face shield or other splatter guard) 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 by the institution; it
should never be taken home by personnel.
- Gloves are worn when hands may
contact potentially infectious materials, contaminated surfaces or
equipment. Wearing two pairs of gloves may be appropriate. Gloves are
disposed of when overtly contaminated, and removed when work with
infectious materials is completed or when the integrity of the glove is
compromised. Disposable gloves are not washed, reused, or used
for touching "clean" surfaces (keyboards, telephones, etc.), and they
should not be worn outside the lab. Alternatives to powdered latex
gloves should be available. Hands are washed following removal of
- Provide lockable doors for
facilities that house restricted agents (as
defined in 42 CFR 72.6).
- Consider locating new
laboratories away from public areas.
- Each laboratory contains a sink
for hand washing. Foot, knee, or automatically operated sinks are
- The laboratory is designed so
that it can be easily cleaned. Carpets and rugs in laboratories are
- Bench tops are impervious to
water and are resistant to moderate heat and the organic solvents,
acids, alkalies, and chemicals used to decontaminate the work surfaces
- 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.
- Install biological safety
cabinets in such a manner that fluctuations of the room supply and
exhaust air do not cause the biological safety cabinets to operate
outside their parameters for containment. Locate biological safety
cabinets away from doors, from windows that can be opened, from heavily
traveled laboratory areas, and from other potentially disruptive
equipment so as to maintain the biological safety cabinets' air flow
parameters for containment.
- An eyewash station is readily
- Illumination is adequate for all
activities, avoiding reflections and glare that could impede vision.
- There are no specific
ventilation requirements. 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. If the
laboratory has windows that open to the exterior, they are fitted with
Reproduced from "Biosafety in Microbiological and Biomedical
Laboratories, BMBL 4th Edition" with permission from the Center for
Disease Control (CDC).