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

 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.
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.

44 Biosafety in Microbiological and Biomedical Laboratories

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.
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.

Section IV—Laboratory Biosafety Level Criteria 45

 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.
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. All persons entering the laboratory are advised of the potential hazards
and meet specific entry/exit requirements in accordance with institutional
policies. Only persons whose presence in the facility or laboratory areas
is required for scientific or support purposes are authorized to enter.
2. All persons who enter operational laboratory areas are provided
information on signs and symptoms of disease and receive occupational
medical services including medical evaluation, surveillance, and
treatment, as appropriate, and offered available immunizations for
agents handled or potentially present in the laboratory.

46 Biosafety in Microbiological and Biomedical Laboratories

3. The laboratory supervisor is responsible for ensuring that laboratory
personnel demonstrate proficiency in standard microbiological practices
and techniques for working with agents requiring BSL-3 containment.
4. A system is established for reporting and documenting near misses,
laboratory accidents, exposures, unanticipated absences due to potential
Laboratory-associated infection, and for the medical surveillance of
potential laboratory-associated illnesses.
5. Incidents that result in exposure to infectious materials are immediately
evaluated per institutional policy. All such incidents are reported to the
laboratory supervisor, institutional management, and appropriate safety,
compliance, and security personnel according to institutional policy.
Appropriate records are maintained.
6. Biological materials that require BSL-3 containment are placed in a
durable leak-proof sealed primary container and then enclosed in a
non-breakable, sealed secondary container prior to removal from the
laboratory. Once removed, the primary container is opened within a BSC
in BSL-3 containment unless a validated inactivation method is used.
See Appendix K. The inactivation method is documented in-house with
viability testing data to support the method.
7. All procedures involving the manipulation of infectious materials are
conducted within a BSC or other physical containment device, when
possible. No work with open vessels is conducted on the bench. If it
is not possible to perform a procedure within a BSC or other physical
containment device, a combination of personal protective equipment
and other administrative and/or engineering controls, such as centrifuge
safety cups or sealed rotors, are used, based on a risk assessment.
Loading and unloading of the rotors and centrifuge safety cups take
place in the BSC or another containment device.
8. Laboratory equipment is routinely decontaminated after spills, splashes,
or other potential contamination, and before repair, maintenance, or
removal from the laboratory.
a. Equipment or material that might be damaged by high temperatures
or steam is decontaminated using an effective and verified method,
such as a gaseous or vapor method.
9. A method for decontaminating all laboratory waste is available in the
facility, preferably within the laboratory (e.g., autoclave, chemical disin-
fection, or other validated decontamination method).

Section IV—Laboratory Biosafety Level Criteria 47

 10. Decontamination of the entire laboratory is considered when there has
been gross contamination of the space, significant changes in laboratory
usage, major renovations, or maintenance shutdowns. Selection of the
appropriate materials and methods used to decontaminate the laboratory
is based on a risk assessment.
11. Decontamination processes are verified on a routine basis.

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

1. Laboratory workers wear protective clothing with a solid-front, such as
tie-back or wrap-around gowns, scrub suits, or coveralls. Protective
clothing is not worn outside of the laboratory. Reusable clothing is
decontaminated before being laundered. Clothing is changed when
contaminated.
2. Based on work being performed, additional PPE may be required.
a. Eye protection and face protection (e.g., safety glasses, goggles,
mask, face shield or other splash 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 decontami-
nated after use.
b. Two pairs of gloves are worn when appropriate.
c. Respiratory protection is considered. Staff wearing respiratory
protection are enrolled in a properly constituted respiratory
protection program.
d. Shoe covers are considered.
3. 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.

D. Laboratory Facilities (Secondary Barriers)

1. The laboratory is separated from areas that are open to unrestricted
traffic flow within the building.
a. Laboratory access is restricted. Laboratory doors are lockable in
accordance with institutional policies. Access to the laboratory is
through two consecutive self-closing doors. A clothing change room
and/or an anteroom may be included in the passageway between
the two self-closing doors.
2. Laboratories have a sink for handwashing. The sink is hands-free
or automatically operated and should be located near the exit door.

48 Biosafety in Microbiological and Biomedical Laboratories

 If a laboratory suite is segregated into different zones, a sink is also
available for handwashing in each zone.
3. An eyewash station is readily available in the laboratory.
4. The laboratory is designed, constructed, and maintained to facilitate
cleaning, decontamination, and housekeeping.
a. Carpets and rugs are not permitted.
b. Spaces between benches, cabinets, and equipment are accessible
for cleaning.
c. Seams, floors, walls, and ceiling surfaces are sealed. Spaces
around doors and ventilation openings are capable of being sealed
to facilitate space decontamination.
d. Floors are slip-resistant, impervious to liquids, and resistant to
chemicals. Flooring is seamless, sealed, or poured with integral
cove bases.
e. Walls and ceilings are constructed to produce a sealed smooth
finish that can be easily cleaned and decontaminated.
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 an
appropriate disinfectant.
6. All windows in the laboratory are sealed.
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. Vacuum lines not protected as described are
capped. The placement of an additional HEPA filter immediately prior to
a central vacuum pump is considered.
9. A ducted mechanical air ventilation system is required. This system
provides sustained directional airflow by drawing air into the laboratory
from “clean” areas toward “potentially contaminated” areas. The
laboratory is designed such that under failure conditions the airflow will
not be reversed at the containment barrier.

Section IV—Laboratory Biosafety Level Criteria 49

 a. A visual monitoring device that confirms directional airflow is
provided at the laboratory entry. Audible alarms to notify personnel
of airflow disruption are considered.
b. The laboratory exhaust air is not re-circulated to any other area in
the building.
c. The laboratory exhaust air is dispersed away from occupied areas
and from building air intake locations or the exhaust air is HEPA
filtered.
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, heavily traveled laboratory areas, and other
possible airflow disruptions.
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.
d. Class III BSCs are provided supply air in such a manner that
prevents positive pressurization of the cabinet or the room.
11. Equipment that may produce infectious aerosols is used within primary
barrier devices that exhaust air through HEPA filtration or other equiv-
alent technology before being discharged into the laboratory. These
HEPA filters are tested annually and replaced as needed.
12. Facility is constructed to allow decontamination of the entire laboratory
when there has been gross contamination of the space, significant
changes in usage, major renovations, or maintenance shutdowns.
Selection of the appropriate materials and methods used to decontam-
inate the laboratory is based on the risk assessment.
a. Facility design consideration is given to means of decontaminating
large pieces of equipment before removal from the laboratory.
13. Enhanced environmental and personal protection may be necessary
based on risk assessment and applicable local, state, or federal
regulations. These laboratory enhancements may include one or more of
the following: an anteroom for clean storage of equipment and supplies

50 Biosafety in Microbiological and Biomedical Laboratories

 with dress-in, shower-out capabilities; gas-tight dampers to facilitate
laboratory isolation; final HEPA filtration of the laboratory exhaust air;
laboratory effluent decontamination; containment of other piped services;
or advanced access control devices, such as biometrics.
14. When present, HEPA filter housings have gas-tight isolation dampers,
decontamination ports, and/or bag-in/bag-out (with appropriate decon-
tamination procedures) capability. All HEPA filters are located as near
as practicable to the laboratory to minimize the length of potentially
contaminated ductwork. The HEPA filter housings allow for leak testing
of each filter and assembly. The filters and housings are certified at least
annually.
15. The BSL-3 facility design, operational parameters, and procedures are
verified and documented prior to operation. Facilities are tested annually
or after significant modification to ensure operational parameters are
met. Verification criteria are modified as necessary by operational
experience.
16. Appropriate communication systems are provided between the
laboratory and the outside (e.g., voice, fax, and computer). Provisions
for emergency communication and emergency access or egress are
developed and implemented.

Biosafety Level 4
Biosafety Level 4 (BSL-4) is required for work with dangerous and exotic agents
that pose a high individual risk of aerosol-transmitted laboratory infections and
life-threatening diseases that are frequently fatal, agents for which there are no
vaccines or treatments, or work with a related agent with unknown risk of trans-
mission. Agents with a close or identical antigenic relationship to agents requiring
BSL-4 containment are handled at this level until sufficient data are obtained to
re-designate the level. Laboratory staff receive specific and thorough training in
handling extremely hazardous infectious agents. Laboratory staff understand the
primary and secondary containment functions of standard and special practices,
containment equipment, and laboratory design characteristics. All laboratory
staff and supervisors are competent in handling agents and procedures requiring
BSL-4 containment. The laboratory supervisor controls access to the laboratory
in accordance with institutional policies.

There are two models for BSL-4 laboratories:

1. Cabinet Laboratory: manipulation of agents is performed in a Class III
BSC; and
2. Suit Laboratory: personnel wear a positive-pressure supplied-air
protective suit.

Section IV—Laboratory Biosafety Level Criteria 51