Biosafety Level 2

Biosafety Level 2 (BSL-2) builds upon BSL-1. BSL-2 is suitable for work with
agents associated with human disease and pose moderate hazards to personnel
and the environment. BSL-2 differs from BSL-1 primarily because: 1) laboratory
personnel receive 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
specifications are recommended for BSL-2.

A. Standard Microbiological Practices

1. The laboratory supervisor enforces the institutional policies that control
safety in and access to the laboratory.
2. The laboratory supervisor ensures that laboratory personnel receive
appropriate training regarding their duties, potential hazards, manipula-
tions of infectious agents, necessary precautions to minimize exposures,
and hazard/exposure evaluation procedures (e.g., physical hazards,
splashes, aerosolization) and that appropriate records are maintained.
Personnel receive annual updates and additional training when
equipment, procedures, or policies change. All persons entering the
facility are advised of the potential hazards, are instructed on the appro-
priate safeguards, and read and follow instructions on practices and
procedures. An institutional policy regarding visitor training, occupational
health requirements, and safety communication is considered.
3. Personal health status may affect an individual’s susceptibility to
infection and ability to receive available immunizations or prophylactic
interventions. Therefore, all personnel, and particularly those of
reproductive age and/or those having conditions that may predispose
them to increased risk for infection (e.g., organ transplant, medical
immunosuppressive agents), are provided information regarding immune
competence and susceptibility to infectious agents. Individuals having
such conditions are encouraged to self-identify to the institution’s
healthcare provider for appropriate counseling and guidance. See
Section VII.
4. A safety manual specific to the facility is prepared or adopted in consul-
tation with the facility director and appropriate safety professionals. The
safety manual is available, accessible, and periodically reviewed and
updated as necessary.

Section IV—Laboratory Biosafety Level Criteria 37

 a. The safety manual contains sufficient information to describe the
biosafety and containment procedures for the organisms and
biological materials in use, appropriate agent-specific decontami-
nation methods, and the work performed.
b. The safety manual contains or references protocols for emergency
situations, including exposures, medical emergencies, facility
malfunctions, and other potential emergencies. Training in
emergency response procedures is provided to emergency
response personnel and other responsible staff according to institu-
tional policies.
5. A sign incorporating the universal biohazard symbol is posted at the
entrance to the laboratory when infectious materials are present. Posted
information includes: the laboratory’s Biosafety Level, the supervisor’s
or other responsible personnel’s name and telephone number, PPE
requirements, general occupational health requirements (e.g., immuniza-
tions, respiratory protection), and required procedures for entering and
exiting the laboratory. Agent information is posted in accordance with the
institutional policy.
6. Long hair is restrained so that it cannot contact hands, specimens,
containers, or equipment.
7. Gloves are worn to protect hands from exposure to hazardous materials.
a. Glove selection is based on an appropriate risk assessment.
b. Gloves are not worn outside the laboratory.
c. Change gloves when contaminated, glove integrity is compromised,
or when otherwise necessary.
d. Do not wash or reuse disposable gloves, and dispose of used
gloves with other contaminated laboratory waste.
8. Gloves and other PPE are removed in a manner that minimizes personal
contamination and transfer of infectious materials outside of the areas
where infectious materials and/or animals are housed or manipulated.
9. Persons wash their hands after working with potentially hazardous
materials and before leaving the laboratory.
10. Eating, drinking, smoking, handling contact lenses, applying cosmetics,
and storing food for human consumption are not permitted in laboratory
areas. Food is stored outside the laboratory area.
11. Mouth pipetting is prohibited. Mechanical pipetting devices are used.

38 Biosafety in Microbiological and Biomedical Laboratories

12. Policies for the safe handling of sharps, such as needles, scalpels,
pipettes, and broken glassware are developed, implemented, and
followed; policies are consistent with applicable state, federal, and
local requirements. Whenever practical, laboratory supervisors adopt
improved engineering and work practice controls that reduce risk of
sharps injuries. Precautions are always taken with sharp items. These
include:
a. Plasticware is substituted for glassware whenever possible.
b. Use of needles and syringes or other sharp instruments is limited
in the laboratory and is restricted to situations where there is no
alternative (e.g., parenteral injection, blood collection, or aspiration
of fluids from laboratory animals or diaphragm bottles). Active or
passive needle-based safety devices are to be used whenever
possible.
i. Uncapping of needles is performed in such a manner to reduce
the potential for recoil causing an accidental needlestick.
ii. Needles are not bent, sheared, broken, recapped, removed
from disposable syringes, or otherwise manipulated by hand
before disposal.
iii. If absolutely necessary to remove a needle from a syringe
(e.g., to prevent lysing blood cells) or recap a needle (e.g.,
loading syringes in one room and injecting animals in another),
a hands-free device or comparable safety procedure must be
used (e.g., a needle remover on a sharps container, the use of
forceps to hold the cap when recapping a needle).
iv. Used, disposable needles and syringes are carefully placed
in puncture-resistant containers used for sharps disposal
immediately after use. The sharps disposal container is located
as close to the point of use as possible.
c. Non-disposable sharps are placed in a hard-walled container for
transport to a processing area for decontamination, preferably by
autoclaving.
d. Broken glassware is not handled directly. Instead, it is removed
using a brush and dustpan, tongs, or forceps.
13. Perform all procedures to minimize the creation of splashes and/or
aerosols.

Section IV—Laboratory Biosafety Level Criteria 39

 14. Decontaminate work surfaces after completion of work and after any spill
or splash of potentially infectious material with appropriate disinfectant.
Spills involving infectious materials are contained, decontaminated, and
cleaned up by staff who are properly trained and equipped to work with
infectious material. A spill procedure is developed and posted within the
laboratory.
15. Decontaminate all cultures, stocks, and other potentially infectious
materials before disposal using an effective method, consistent with
applicable institutional, local, and state requirements. Depending on
where the decontamination will be performed, the following methods are
used prior to transport:
a. Materials to be decontaminated outside of the immediate laboratory
are placed in a durable, leak-proof container and secured for
transport. For infectious materials, the outer surface of the container
is disinfected prior to moving materials and the transport container
has a universal biohazard label.
b. Materials to be removed from the facility for decontamination are
packed in accordance with applicable local, state, and federal
regulations.
16. An effective integrated pest management program is implemented. See
Appendix G.
17. Animals and plants not associated with the work being performed are not
permitted in the laboratory.

B. Special Practices
1. Access to the laboratory is controlled when work is being conducted.
2. The laboratory supervisor is responsible for ensuring that laboratory
personnel demonstrate proficiency in standard microbiological practices
and techniques for working with agents requiring BSL-2 containment.
3. Laboratory personnel are provided medical surveillance, as appropriate,
and offered available immunizations for agents handled or potentially
present in the laboratory.
4. Properly maintained BSCs or other physical containment devices are
used, when possible, whenever:
a. Procedures with a potential for creating infectious aerosols or
splashes are conducted. These include pipetting, centrifuging,
grinding, blending, shaking, mixing, sonicating, opening containers
of infectious materials, inoculating animals intranasally, and
harvesting infected tissues from animals or eggs.

40 Biosafety in Microbiological and Biomedical Laboratories

 b. High concentrations or large volumes of infectious agents are used.
Such materials may be centrifuged in the open laboratory using
sealed rotors or centrifuge safety cups with loading and unloading
of the rotors and centrifuge safety cups in the BSC or another
containment device.
c. If it is not possible to perform a procedure within a BSC or other
physical containment device, a combination of appropriate personal
protective equipment and administrative controls are used, based on
a risk assessment.
5. Laboratory equipment is decontaminated routinely; after spills, splashes,
or other potential contamination; and before repair, maintenance, or
removal from the laboratory.
6. A method for decontaminating all laboratory waste is available (e.g.,
autoclave, chemical disinfection, incineration, or other validated decon-
tamination method).
7. Incidents that may result in exposure to infectious materials are immedi-
ately evaluated per institutional policies. All such incidents are reported
to the laboratory supervisor and any other personnel designated by the
institution. Appropriate records are maintained.

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

1. Protective laboratory coats, gowns, or uniforms designated for laboratory
use are worn while working with hazardous materials and removed
before leaving for non-laboratory areas (e.g., cafeteria, library, and
administrative offices). Protective clothing is disposed of appropriately or
deposited for laundering by the institution. Laboratory clothing is not
taken home.

2. Eye protection and face protection (e.g., safety glasses, goggles, mask,
face shield or other splatter guard) are used for manipulations or activities
that may result in splashes or sprays of infectious or other hazardous
materials. Eye protection and face protection are disposed of with other
contaminated laboratory waste or decontaminated after use.
3. The risk assessment considers whether respiratory protection is needed
for the work with hazardous materials. If needed, relevant staff are
enrolled in a properly constituted respiratory protection program.
4. In circumstances where research animals are present in the laboratory,
the risk assessment considers appropriate eye, face, and respiratory
protection, as well as potential animal allergens.

Section IV—Laboratory Biosafety Level Criteria 41

D. Laboratory Facilities (Secondary Barriers)
1. Laboratory doors are self-closing and have locks in accordance with the
institutional policies.
2. Laboratories have a sink for handwashing. It should be located near the
exit door.
3. An eyewash station is readily available in the laboratory.
4. The laboratory is designed so that it can be easily cleaned.
a. Carpets and rugs in laboratories are not appropriate.
b. Spaces between benches, cabinets, and equipment are accessible
for cleaning.
5. Laboratory furniture can support anticipated loads and uses.
a. Benchtops are impervious to water and resistant to heat, organic
solvents, acids, alkalis, and other chemicals.
b. Chairs used in laboratory work are covered with a non-porous
material that can be easily cleaned and decontaminated with appro-
priate disinfectant.
6. Laboratory windows that open to the exterior are not recommended.
However, if a laboratory does have windows that open to the exterior,
they are fitted with screens.
7. Illumination is adequate for all activities and avoids reflections and glare
that could impede vision.
8. Vacuum lines in use are protected with liquid disinfectant traps and
in-line HEPA filters or their equivalent. See Appendix A, Figure 11. Filters
are replaced, as needed, or are on a replacement schedule determined
by a risk assessment.
9. There are no specific requirements for ventilation systems. However, the
planning of new facilities considers mechanical ventilation systems that
provide an inward flow of air without recirculation to spaces outside of
the laboratory.
10. BSCs and other primary containment barrier systems are installed and
operated in a manner to ensure their effectiveness. See Appendix A.
a. BSCs are installed so that fluctuations of the room air supply and
exhaust do not interfere with proper operations. BSCs are located
away from doors, windows that can be opened, heavily traveled
laboratory areas, and other possible airflow disruptions.

42 Biosafety in Microbiological and Biomedical Laboratories

 b. BSCs can be connected to the laboratory exhaust system by either
a canopy connection (Class IIA only) or directly exhausted to the
outside through a hard connection (Class IIB, IIC, or III). Class IIA or
IIC BSC exhaust can be safely recirculated back into the laboratory
environment if no volatile toxic chemicals are used in the cabinet.
c. BSCs are certified at least annually to ensure correct performance,
or as specified in Appendix A, Part 7.

Biosafety Level 3
Biosafety Level 3 (BSL-3) is suitable for work with indigenous or exotic agents
that may cause serious or potentially lethal disease through the inhalation route
of exposure. Laboratory personnel receive specific training in handling pathogenic
and potentially lethal agents, and they are supervised by scientists competent in
handling infectious agents and associated procedures.

A BSL-3 laboratory has special engineering and design features.

The following standard and special practices, safety equipment, and facility
specifications are recommended for BSL-3.

A. Standard Microbiological Practices

1. The laboratory supervisor enforces the institutional policies that control
safety in and access to the laboratory.
2. The laboratory supervisor ensures that laboratory personnel receive
appropriate training regarding their duties, potential hazards, manipula-
tions of infectious agents, necessary precautions to minimize exposures,
and hazard/exposure evaluation procedures (e.g., physical hazards,
splashes, aerosolization) and that appropriate records are maintained.
Personnel receive annual updates and additional training when
equipment, procedures, or policies change. All persons entering the
facility are advised of the potential hazards, are instructed on the appro-
priate safeguards, and read and follow instructions on practices and
procedures. An institutional policy regarding visitor training, occupational
health requirements, and safety communication is considered.
3. Personal health status may affect an individual’s susceptibility to
infection and ability to receive available immunizations or prophylactic
interventions. Therefore, all personnel, and particularly those of
reproductive age and/or those having conditions that may predispose
them to increased risk for infection (e.g., organ transplant, medical
immunosuppressive agents), are provided information regarding immune
competence and susceptibility to infectious agents. Individuals having

Section IV—Laboratory Biosafety Level Criteria 43