APPENDIX G.................... PHYSICAL
CONTAINMENT
Appendix
G-I................ Standard Practices and Training
Appendix
G-II................ Physical Containment Levels
Appendix
G-II-A-1.......... Standard Microbiological Practices (BL1)
Appendix
G-II-A-2.......... Special Practices (BL1)
Appendix
G-II-A-3.......... Containment Equipment (BL1)
Appendix
G-II-A-4.......... Laboratory Facilities (BL1)
Appendix
G-II-B-1.......... Standard Microbiological Practices (BL2)
Appendix
G-II-B-2.......... Special Practices (BL2)
Appendix
G-II-B-3.......... Containment Equipment (BL2)
Appendix
G-II-B-4.......... Laboratory Facilities (BL2)
Appendix
G-II-C-1.......... Standard Microbiological Practices (BL3)
Appendix
G-II-C-2.......... Special Practices (BL3)
Appendix
G-II-C-2-t........ Alternative Selection of Containment Equipment
(BL3)
Appendix
G-II-C-3.......... Containment Equipment (BL3)
Appendix
G-II-C-4.......... Laboratory Facilities (BL3)
Appendix
G-II-D............ Biosafety Level 4 (BL4)
Appendix
G-II-D-1.......... Standard Microbiological Practices (BL4)
Appendix
G-II-D-2.......... Special Practices (BL4)
Appendix
G-II-D-2-m...... Alternative Selection of Containment Equipment
(BL4)
Appendix
G-II-D-3.......... Containment Equipment (BL4)
Appendix
G-II-D-4.......... Laboratory Facilities (BL4)
Appendix
G-III............... Footnotes and References of Appendix G
LIST OF TABLES
Appendix G specifies physical containment for standard
laboratory experiments and defines Biosafety Level 1 through Biosafety Level
4. For large-scale (over 10 liters)
research or production, Appendix K (Physical Containment for Large Scale
Uses of Organisms Containing Recombinant DNA Molecules) supersedes Appendix
G. Appendix K defines Good Large Scale Practice through Biosafety
Level 3 - Large Scale. For certain work
with plants, Appendix P (Physical and Biological Containment for Recombinant
DNA Research Involving Plants) supersedes Appendix G. Appendix
P defines Biosafety Levels 1 through 4 - Plants. For certain work with animals, Appendix Q (Physical and
Biological Containment for Recombinant DNA Research Involving Animals)
supersedes Appendix G. Appendix Q defines Biosafety Levels 1
through 4 - Animals.
The first principle of containment is strict adherence to
good microbiological practices (see Appendices
G-III-A through G-III-J, Footnotes and References of Appendix G). Consequently, all personnel directly or
indirectly involved in experiments using recombinant DNA shall receive adequate
instruction (see Sections IV-B-1-h, Responsibilities of the
Institution--General Information, and IV-B-7-d, Responsibilities of the Principal Investigator
Prior to Initiating Research). At a
minimum, these instructions include training in aseptic techniques and in the
biology of the organisms used in the experiments so that the potential
biohazards can be understood and appreciated.
Any research group working with agents that are known or
potential biohazards shall have an emergency plan that describes the procedures
to be followed if an accident contaminates personnel or the environment. The Principal Investigator shall ensure that
everyone in the laboratory is familiar with both the potential hazards of the work
and the emergency plan (see Sections IV-B-7-d, Responsibilities of the Principal
Investigator Prior to Initiating Research and IV-B-7-e, Responsibilities
of the Principal Investigator During the Conduct of the Research). If a research group is working with a known
pathogen for which there is an effective vaccine, the vaccine should be made
available to all workers. Serological
monitoring, when clearly appropriate, will be provided (see Section
IV-B-1-f, Responsibilities of the Institution--General Information).
The Laboratory Safety Monograph (see Appendix G-III-O, Footnotes and
References of Appendix G) and Biosafety in Microbiological and
Biomedical Laboratories (see Appendix
G-III-B, Footnotes and References of Appendix G) describe practices,
equipment, and facilities in detail.
The objective of physical containment is to confine
organisms containing recombinant DNA molecules and to reduce the potential for
exposure of the laboratory worker, persons outside of the laboratory, and the
environment to organisms containing recombinant DNA molecules. Physical containment is achieved through the
use of laboratory practices, containment equipment, and special laboratory
design. Emphasis is placed on primary
means of physical containment which are provided by laboratory practices and
containment equipment. Special
laboratory design provides a secondary means of protection against the
accidental release of organisms outside the laboratory or to the
environment. Special laboratory design
is used primarily in facilities in which experiments of moderate to high
potential hazard are performed.
Combinations of laboratory practices, containment equipment,
and special laboratory design can be made to achieve different levels of
physical containment. Four levels of
physical containment, which are designated as BL1, BL2, BL3, and BL4 are
described. It should be emphasized that
the descriptions and assignments of physical containment detailed below are
based on existing approaches to containment of pathogenic organisms (see Appendix G-III-B, Footnotes and
References of Appendix G). The
National Cancer Institute describes three levels for research on oncogenic
viruses which roughly correspond to our BL2, BL3, and BL4 levels (see Appendix G-III-C, Footnotes and
References of Appendix G).
It is recognized that several different combinations of
laboratory practices, containment equipment, and special laboratory design may
be appropriate for containment of specific research activities. The NIH Guidelines, therefore, allow
alternative selections of primary containment equipment within facilities that
have been designed to provide BL3 and BL4 levels of physical containment. The selection of alternative methods of
primary containment is dependent, however, on the level of biological
containment provided by the host-vector system used in the experiment. Consideration will be given to other
combinations which achieve an equivalent level of containment (see Sections IV-C-1-b-(1),
Major Actions and IV-C-1-b-(2),
Minor Actions).
Appendix G-II-A-1-a.
Access to the laboratory is limited or restricted at the discretion of
the Principal Investigator when experiments are in progress.
Appendix G-II-A-1-b.
Work surfaces are decontaminated once a day and after any spill of
viable material.
Appendix G-II-A-1-c.
All contaminated liquid or solid wastes are decontaminated before
disposal.
Appendix G-II-A-1-d.
Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-A-1-e.
Eating, drinking, smoking, and applying cosmetics are not permitted in
the work area. Food may be stored in
cabinets or refrigerators designated and used for this purpose only.
Appendix G-II-A-1-f.
Persons wash their hands: (i)
after they handle materials involving organisms containing recombinant DNA
molecules and animals, and (ii) before exiting the laboratory.
Appendix G-II-A-1-g.
All procedures are performed carefully to minimize the creation of
aerosols.
Appendix G-II-A-1-h.
In the interest of good personal hygiene, facilities (e.g., hand washing
sink, shower, changing room) and protective clothing (e.g., uniforms,
laboratory coats) shall be provided that are appropriate for the risk of
exposure to viable organisms containing recombinant DNA molecules.
Appendix G-II-A-2-a.
Contaminated materials that are to be decontaminated at a site away from
the laboratory are placed in a durable leak-proof container which is closed
before being removed from the laboratory.
Appendix G-II-A-2-b.
An insect and rodent control program is in effect.
Appendix G-II-A-3-a.
Special containment equipment is generally not required for
manipulations of agents assigned to BL1.
Appendix G-II-A-4-a.
The laboratory is designed so that it can be easily cleaned.
Appendix G-II-A-4-b.
Bench tops are impervious to water and resistant to acids, alkalis,
organic solvents, and moderate heat.
Appendix G-II-A-4-c.
Laboratory furniture is sturdy.
Spaces between benches, cabinets, and equipment are accessible for
cleaning.
Appendix G-II-A-4-d.
Each laboratory contains a sink for hand washing.
Appendix G-II-A-4-e.
If the laboratory has windows that open, they are fitted with fly
screens.
Appendix G-II-B-1-a.
Access to the laboratory is limited or restricted by the Principal Investigator
when work with organisms containing recombinant DNA molecules is in progress.
Appendix G-II-B-1-b.
Work surfaces are decontaminated at least once a day and after any spill
of viable material.
Appendix G-II-B-1-c.
All contaminated liquid or solid wastes are decontaminated before
disposal.
Appendix G-II-B-1-d.
Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-B-1-e.
Eating, drinking, smoking, and applying cosmetics are not permitted in
the work area. Food may be stored in
cabinets or refrigerators designated and used for this purpose only.
Appendix G-II-B-1-f.
Persons wash their hands: (i)
after handling materials involving organisms containing recombinant DNA
molecules and animals, and (ii) when exiting the laboratory.
Appendix G-II-B-1-g.
All procedures are performed carefully to minimize the creation of
aerosols.
Appendix G-II-B-1-h.
Experiments of lesser biohazard potential can be conducted concurrently
in carefully demarcated areas of the same laboratory.
Appendix G-II-B-2-a.
Contaminated materials that are to be decontaminated at a site away from
the laboratory are placed in a durable leak-proof container which is closed
before being removed from the laboratory.
Appendix G-II-B-2-b.
The Principal Investigator limits access to the laboratory. The Principal Investigator has the final
responsibility for assessing each circumstance and determining who may enter or
work in the laboratory.
Appendix G-II-B-2-c.
The Principal Investigator establishes policies and procedures whereby
only persons who have been advised of the potential hazard and meet any
specific entry requirements (e.g., immunization) may enter the laboratory or
animal rooms.
Appendix G-II-B-2-d.
When the organisms containing recombinant DNA molecules in use in the
laboratory require special provisions for entry (e.g., vaccination), a hazard
warning sign incorporating the universal biosafety symbol is posted on the
access door to the laboratory work area.
The hazard warning sign identifies the agent, lists the name and
telephone number of the Principal Investigator or other responsible person(s),
and indicates the special requirement(s) for entering the laboratory.
Appendix G-II-B-2-e. An insect and rodent control program is in effect.
Appendix G-II-B-2-f.
Laboratory coats, gowns, smocks, or uniforms are worn while in the
laboratory. Before exiting the
laboratory for non-laboratory 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.
Appendix G-II-B-2-g.
Animals not involved in the work being performed are not permitted in
the laboratory.
Appendix G-II-B-2-h.
Special care is taken to avoid skin contamination with organisms
containing recombinant DNA molecules; gloves should be worn when handling
experimental animals and when skin contact with the agent is unavoidable.
Appendix G-II-B-2-i.
All wastes from laboratories and animal rooms are appropriately
decontaminated before disposal.
Appendix G-II-B-2-j.
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., needle is integral to the syringe) are used for the
injection or aspiration of fluids containing organisms that contain recombinant
DNA molecules. Extreme caution should
be used when handling needles and syringes to avoid autoinoculation and the
generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the needle
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
autoclaved, before discard or reuse.
Appendix G-II-B-2-k.
Spills and accidents which result in overt exposures to organisms
containing recombinant DNA molecules are immediately reported to the
Institutional Biosafety Committee and NIH/OBA.
Reports to NIH/OBA shall be sent to the Office of Biotechnology
Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC
7985, Bethesda, MD 20892-7985 (20817
for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, surveillance, and treatment are provided as
appropriate and written records are maintained.
Appendix G-II-B-2-l.
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.
Appendix G-II-B-2-m.
A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read
and follow instructions on practices and procedures.
Appendix G-II-B-3-a.
Biological safety cabinets (Class I or II) (see Appendix G-III-L, Footnotes and
References of Appendix G) or other appropriate personal protective or physical
containment devices are used whenever:
Appendix G-II-B-3-a-(1). Procedures with a high potential for creating aerosols are
conducted (see Appendix G-III-O, Footnotes
and References of Appendix G). These
may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic
disruption, opening containers of materials whose internal pressures may be
different from ambient pressures, intranasal inoculation of animals, and
harvesting infected tissues from animals or eggs.
Appendix G-II-B-3-a-(2). High concentrations or large volumes of organisms containing
recombinant DNA molecules are used.
Such materials may be centrifuged in the open laboratory if sealed beads
or centrifuge safety cups are used and if they are opened only in a biological
safety cabinet.
Appendix G-II-B-4-a.
The laboratory is designed so that it can be easily cleaned.
Appendix G-II-B-4-b.
Bench tops are impervious to water and resistant to acids, alkalis,
organic solvents, and moderate heat.
Appendix G-II-B-4-c.
Laboratory furniture is sturdy and spaces between benches, cabinets, and
equipment are accessible for cleaning.
Appendix G-II-B-4-d.
Each laboratory contains a sink for hand washing.
Appendix G-II-B-4-e.
If the laboratory has windows that open, they are fitted with fly
screens.
Appendix G-II-B-4-f.
An autoclave for decontaminating laboratory wastes is available.
Appendix G-II-C-1-a.
Work surfaces are decontaminated at least once a day and after any spill
of viable material.
Appendix G-II-C-1-b.
All contaminated liquid or solid wastes are decontaminated before
disposal.
Appendix G-II-C-1-c.
Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-C-1-d.
Eating, drinking, smoking, storing food, and applying cosmetics are not
permitted in the work area.
Appendix G-II-C-1-e.
Persons wash their hands: (i)
after handling materials involving organisms containing recombinant DNA molecules,
and handling animals, and (ii) when exiting the laboratory.
Appendix G-II-C-1-f.
All procedures are performed carefully to minimize the creation of
aerosols.
Appendix G-II-C-1-g.
Persons under 16 years of age shall not enter the laboratory.
Appendix G-II-C-1-h.
If experiments involving other organisms which require lower levels of
containment are to be conducted in the same laboratory concurrently with
experiments requiring BL3 level physical containment, they shall be conducted
in accordance with all BL3 level laboratory practices.
Appendix G-II-C-2-a.
Laboratory doors are kept closed when experiments are in progress.
Appendix G-II-C-2-b.
Contaminated materials that are to be decontaminated at a site away from
the laboratory are placed in a durable leak-proof container which is closed
before being removed from the laboratory.
Appendix G-II-C-2-c.
The Principal Investigator controls access to the laboratory and
restricts access to persons whose presence is required for program or support
purposes. The Principal Investigator
has the final responsibility for assessing each circumstance and determining
who may enter or work in the laboratory.
Appendix G-II-C-2-d.
The Principal Investigator 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 entering the laboratory or animal rooms.
Appendix G-II-C-2-e.
When organisms containing recombinant DNA molecules or experimental
animals are present in the laboratory or containment module, a hazard warning
sign incorporating the universal biosafety 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 Principal Investigator 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 measures.
Appendix G-II-C-2-f.
All activities involving organisms containing recombinant DNA molecules
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.
Appendix G-II-C-2-g.
The work surfaces of biological safety cabinets and other containment
equipment are decontaminated when work with organisms containing recombinant
DNA molecules is finished.
Plastic-backed paper toweling used on non-perforated work surfaces
within biological safety cabinets facilitates clean-up.
Appendix G-II-C-2-h.
An insect and rodent program is in effect.
Appendix G-II-C-2-i.
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 prior to laundering or disposal.
Appendix G-II-C-2-j.
Special care is taken to avoid skin contamination with contaminated
materials; gloves should be worn when handling infected animals and when skin
contact with infectious materials is unavoidable.
Appendix G-II-C-2-k.
Molded surgical masks or respirators are worn in rooms containing
experimental animals.
Appendix G-II-C-2-l.
Animals and plants not related to the work being conducted are not
permitted in the laboratory.
Appendix G-II-C-2-m.
Laboratory animals held in a BL3 area shall be housed in partial-containment
caging systems, such as Horsfall units (see Appendix G-III-K, Footnotes and
References of Appendix G), open cages placed in ventilated enclosures,
solid-wall and -bottom cages covered by filter bonnets or solid-wall and
-bottom cages placed on holding racks equipped with ultraviolet in radiation
lamps and reflectors.
Note:
Conventional caging systems may be used provided that all personnel wear
appropriate personal protective devices.
These protective devices shall include at a minimum wrap-around gowns,
head covers, gloves, shoe covers, and respirators. All personnel shall shower on exit from areas where these devices
are required.
Appendix G-II-C-2-n.
All wastes from laboratories and animal rooms are appropriately
decontaminated before disposal.
Appendix G-II-C-2-o.
Vacuum lines are protected with high efficiency particulate air/HEPA
filters and liquid disinfectant traps.
Appendix G-II-C-2-p.
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., needle is integral to the syringe) are used for the
injection or aspiration of fluids containing organisms that contain recombinant
DNA molecules. Extreme caution should
be used when handling needles and syringes to avoid autoinoculation and the
generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the needle
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.
Appendix G-II-C-2-q.
Spills and accidents which result in overt or potential exposures to
organisms containing recombinant DNA molecules are immediately reported to the
Biological Safety Officer, Institutional Biosafety Committee, and NIH/OBA. Reports to NIH/OBA shall be sent to the
Office of Biotechnology Activities, National Institutes of Health, 6705
Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839
(fax). Appropriate medical evaluation,
surveillance, and treatment are provided and written records are maintained.
Appendix G-II-C-2-r.
Baseline serum samples for all laboratory and other at-risk personnel
should be collected and stored.
Additional serum specimens may be collected periodically depending on
the agents handled or the function of the laboratory.
Appendix G-II-C-2-s.
A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read
and follow the instructions on practices and procedures.
Experimental procedures involving a host-vector system that
provides a one-step higher level of biological containment than that specified
may be conducted in the BL3 laboratory using containment equipment specified
for the BL2 level of physical containment.
Experimental procedures involving a host-vector system that provides a
one-step lower level of biological containment than that specified may be
conducted in the BL3 laboratory using containment equipment specified for the
BL4 level of physical containment.
Alternative combination of containment safeguards are shown in Appendix G-Table 1, Possible
Alternate Combinations of Physical and Biological Containment Safeguards.
Appendix G-II-C-3-a.
Biological safety cabinets (Class I, II, or III) (see Appendix G-III-L, Footnotes and
References of Appendix G) or other appropriate combinations of personal
protective 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 organisms
containing recombinant DNA molecules which pose a threat of aerosol
exposure. These include: manipulation of cultures and of those
clinical or environmental materials which may be a source of aerosols; the
aerosol challenge of experimental animals; the harvesting of infected tissues
or fluids from experimental animals and embryonate eggs; and the necropsy of
experimental animals.
Appendix G-II-C-4-a.
The laboratory is separated from areas which 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 be
provided by a double-doored clothes change room (showers may be included),
airlock, or other access facility which requires passage through two sets of
doors before entering the laboratory.
Appendix G-II-C-4-b.
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.
Appendix G-II-C-4-c.
Bench tops are impervious to water and resistant to acids, alkalis,
organic solvents, and moderate heat.
Appendix G-II-C-4-d.
Laboratory furniture is sturdy and spaces between benches, cabinets, and
equipment are accessible for cleaning.
Appendix G-II-C-4-e.
Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically
operated and is located near the laboratory exit door.
Appendix G-II-C-4-f.
Windows in the laboratory are closed and sealed.
Appendix G-II-C-4-g.
Access doors to the laboratory or containment module are self-closing.
Appendix G-II-C-4-h.
An autoclave for decontaminating laboratory wastes is available
preferably within the laboratory.
Appendix G-II-C-4-i.
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 the occupied
areas and air intakes. Personnel shall
verify that the direction of the airflow (into the laboratory) is proper. The exhaust air from the laboratory room may
be discharged to the outside without being filtered or otherwise treated.
Appendix G-II-C-4-j.
The high efficiency particulate air/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 twelve 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 air system, it is
connected to this system in a manner (e.g., thimble unit connection (see Appendix G-III-L, Footnotes and
References of Appendix G)) that avoids any interference with the air
balance of the cabinets or building exhaust system.
Appendix G-II-D-1-a.
Work surfaces are decontaminated at least once a day and immediately
after any spill of viable material.
Appendix G-II-D-1-b.
Only mechanical pipetting devices are used.
Appendix G-II-D-1-c.
Eating, drinking, smoking, storing food, and applying cosmetics are not
permitted in the laboratory.
Appendix G-II-D-1-d.
All procedures are performed carefully to minimize the creation of
aerosols.
Appendix G-II-D-2-a.
Biological materials to be removed from the Class III cabinets or from
the maximum containment laboratory in a viable or intact state are transferred
to a non-breakable, sealed primary container and then enclosed in a
non-breakable, sealed secondary container which is removed from the facility
through a disinfectant dunk tank, fumigation chamber, or an airlock designed
for this purpose.
Appendix G-II-D-2-b.
No materials, except for biological materials that are to remain in a
viable or intact state, are removed from the maximum containment laboratory
unless they have been autoclaved or decontaminated before exiting the
facility. Equipment or material which
might be damaged by high temperatures or steam is decontaminated by gaseous or
vapor methods in an airlock or chamber designed for this purpose.
Appendix G-II-D-2-c.
Only persons whose presence in the facility or individual laboratory
rooms is required for program or support purposes are authorized to enter. The supervisor has the final responsibility
for assessing each circumstance and determining who may enter or work in the
laboratory. Access to the facility is
limited by means of secure, locked doors; accessibility is managed by the
Principal Investigator, Biological Safety Officer, or other person responsible
for the physical security of the facility.
Before entering, persons are advised of the potential biohazards and
instructed as to appropriate safeguards for ensuring their safety. Authorized persons comply with the
instructions and all other applicable entry and exit procedures. A logbook signed by all personnel indicates
the date and time of each entry and exit.
Practical and effective protocols for emergency situations are
established.
Appendix G-II-D-2-d.
Personnel enter and exit the facility only through the clothing change
and shower rooms. Personnel shower each
time they exit the facility. Personnel
use the air locks to enter or exit the laboratory only in an emergency.
Appendix G-II-D-2-e.
Street clothing is removed in the outer clothing change room and kept
there. Complete laboratory clothing
(may be disposable), including undergarments, pants and shirts or jump suits,
shoes, and gloves, is provided and used by all personnel entering the facility.
Head covers are provided for personnel
who do not wash their hair during the exit shower. When exiting the laboratory and before proceeding into the shower
area, personnel remove their laboratory clothing and store it in a locker or
hamper in the inner change room.
Protective clothing shall be decontaminated prior to laundering or
disposal.
Appendix G-II-D-2-f.
When materials that contain organisms containing recombinant DNA
molecules or experimental animals are present in the laboratory or animal rooms,
a hazard warning sign incorporating the universal biosafety symbol is posted on
all access doors. The sign identifies
the agent, lists the name of the Principal Investigator or other responsible
person(s), and indicates any special requirements for entering the area (e.g.,
the need for immunizations or respirators).
Appendix G-II-D-2-g.
Supplies and materials needed in the facility are brought in by way of
the double-doored autoclave, fumigation chamber, or airlock which is
appropriately decontaminated between each use.
After securing the outer doors, personnel within the facility retrieve
the materials by opening the interior doors or the autoclave, fumigation
chamber, or airlock. These doors are
secured after materials are brought into the facility.
Appendix G-II-D-2-h.
An insect and rodent control program is in effect.
Appendix G-II-D-2-i.
Materials (e.g., plants, animals, and clothing) not related to the
experiment being conducted are not permitted in the facility.
Appendix G-II-D-2-j.
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., needle is integral part of unit) are used for the
injection or aspiration of fluids containing organisms that contain recombinant
DNA molecules. Needles should not be
bent, sheared, replaced in the needle sheath or guard, or removed from the
syringe following use. The needle and
syringe should be placed in a puncture-resistant container and decontaminated,
preferably by autoclaving before discard or reuse. Whenever possible, cannulas are used instead of sharp needles
(e.g., gavage).
Appendix G-II-D-2-k.
A system is set up for reporting laboratory accidents, exposures,
employee absenteeism, and for the medical surveillance of potential
laboratory-associated illnesses. Spills
and accidents which result in overt exposures to organisms containing
recombinant DNA molecules are immediately reported to the Biological Safety
Officer, Institutional Biosafety Committee, and NIH/OBA. Reports to the NIH/OBA shall be sent to the
Office of Biotechnology Activities, National Institutes of Health, 6705
Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839
(fax). Written records are prepared and
maintained. An essential adjunct to
such a reporting-surveillance system is the availability of a facility for
quarantine, isolation, and medical care of personnel with potential or known
laboratory associated illnesses.
Appendix G-II-D-2-l.
Laboratory animals involved in experiments requiring BL4 level physical
containment shall be housed either in cages contained in Class III cabinets or
in partial containment caging systems, such as Horsfall units (see Appendix G-III-K, Footnotes and
References of Appendix G), open cages placed in ventilated enclosures, or
solid-wall and -bottom cages placed on holding racks equipped with ultraviolet
irradiation lamps and reflectors that are located in a specially designed area
in which all personnel are required to wear one-piece positive pressure suits.
Experimental procedures involving a host-vector system that
provides a one-step higher level of biological containment than that specified
may be conducted in the BL4 facility using containment equipment requirements
specified for the BL3 level of physical containment. Alternative combinations of containment safeguards are shown in
Appendix G-Table 1, Possible Alternate Combinations of Physical and
Biological Containment Safeguards.
Appendix G-II-D-3-a.
All procedures within the facility with agents assigned to Biosafety
Level 4 are conducted in the Class III biological safety cabinet or in Class I
or II biological safety cabinets used in conjunction with one-piece positive
pressure personnel suits ventilated by a life-support system.
Appendix G-II-D-4-a.
The maximum containment facility consists of either a separate building
or a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a
shower are provided for personnel entering and exiting the facility. A double-doored autoclave, fumigation
chamber, or ventilated airlock is provided for passage of those materials,
supplies, or equipment which are not brought into the facility through the
change room.
Appendix G-II-D-4-b.
Walls, floors, and ceilings of the facility are constructed to form a
sealed internal shell which facilitates fumigation and is animal and insect
proof. The internal surfaces of this
shell are resistant to liquids and chemicals, thus facilitating cleaning and
decontamination of the area. All
penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps
filled with a chemical disinfectant of demonstrated efficacy against the target
agent, and they are connected directly to the liquid waste decontamination
system. Sewer and other ventilation
lines contain high efficiency particulate air/HEPA filters.
Appendix G-II-D-4-c.
Internal facility appurtenances, such as light fixtures, air ducts, and
utility pipes, are arranged to minimize the horizontal surface area on which
dust can settle.
Appendix G-II-D-4-d.
Bench tops have seamless surfaces which are impervious to water and
resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix G-II-D-4-e.
Laboratory furniture is simple and of sturdy construction; and spaces
between benches, cabinets, and equipment are accessible for cleaning.
Appendix G-II-D-4-f.
A foot, elbow, or automatically operated hand washing sink is provided
near the door of each laboratory room in the facility.
Appendix G-II-D-4-g.
If there is a central vacuum system, it does not serve areas outside the
facility. In-line high efficiency
particulate air/HEPA filters are placed as near as practicable to each use
point or service cock. Filters are
installed to permit in-place decontamination and replacement. Other liquid and gas services to the
facility are protected by devices that prevent back-flow.
Appendix G-II-D-4-h.
If water fountains are provided, they are foot operated and are located
in the facility corridors outside the laboratory. The water service to the fountain is not connected to the
back-flow protected distribution system supplying water to the laboratory
areas.
Appendix G-II-D-4-i.
Access doors to the laboratory are self-closing and locking.
Appendix G-II-D-4-j.
Any windows are breakage resistant.
Appendix G-II-D-4-k.
A double-doored autoclave is provided for decontaminating materials
passing out of the facility. The
autoclave door which opens to the area external to the facility is sealed to
the outer wall and automatically controlled so that the outside door can only
be opened after the autoclave "sterilization" cycle has been
completed.
Appendix G-II-D-4-l.
A pass-through dunk tank, fumigation chamber, or an equivalent
decontamination method is provided so that materials and equipment that cannot
be decontaminated in the autoclave can be safely removed from the facility.
Appendix G-II-D-4-m.
Liquid effluent from laboratory sinks, biological safety cabinets,
floors, and autoclave chambers are decontaminated by heat treatment before
being released from the maximum containment facility. Liquid wastes from shower rooms and toilets may be decontaminated
with chemical disinfectants or by heat in the liquid waste decontamination
system. The procedure used for heat
decontamination of liquid wastes is evaluated mechanically and biologically by
using a recording thermometer and an indicator microorganism with a defined
heat susceptibility pattern. If liquid
wastes from the shower room are decontaminated with chemical disinfectants, the
chemical used is of demonstrated efficacy against the target or indicator
microorganisms.
Appendix G-II-D-4-n.
An individual supply and exhaust air ventilation system is
provided. The system maintains pressure
differentials and directional airflow as required to assure flows inward from
areas outside of the facility toward areas of highest potential risk within the
facility. Manometers are used to sense
pressure differentials between adjacent areas maintained at different pressure
levels. If a system malfunctions, the
manometers sound an alarm. The supply
and exhaust airflow is interlocked to assure inward (or zero) airflow at all
times.
Appendix G-II-D-4-o.
The exhaust air from the facility is filtered through high efficiency particulate
air/HEPA filters and discharged to the outside so that it is dispersed away
from occupied buildings and air intakes.
Within the facility, the filters are located as near the laboratories as
practicable in order to reduce the length of potentially contaminated air
ducts. The filter chambers are designed
to allow in situ decontamination before filters are removed and to
facilitate certification testing after they are replaced. Coarse filters and HEPA filters are provided
to treat air supplied to the facility in order to increase the lifetime of the
exhaust HEPA filters and to protect the supply air system should air pressures
become unbalanced in the laboratory.
Appendix G-II-D-4-p.
The treated exhaust air from Class I and II biological safety cabinets
may be discharged into the laboratory room environment or the outside through
the facility air exhaust system. If
exhaust air from Class I or II biological safety cabinets is discharged into
the laboratory the cabinets are tested and certified at six-month
intervals. The exhaust air from
Class III biological safety cabinets is discharged, without recirculation
through two sets of high efficiency particulate air/HEPA filters in series, via
the facility exhaust air system. If
the treated exhaust air from any of these cabinets is discharged to the outside
through the facility exhaust air system, it is connected to this system in a
manner (e.g., thimble unit connection (see Appendix
G-III-L, Footnotes and References of Appendix G)) that avoids any
interference with the air balance of the cabinets or the facility exhaust air
system.
Appendix G-II-D-4-q.
A specially designed suit area may be provided in the facility. Personnel who enter this area shall wear a
one-piece positive pressure suit that is ventilated by a life-support
system. The life-support system
includes alarms and emergency backup breathing air tanks. Entry to this area is through an airlock
fitted with airtight doors. A chemical
shower is provided to decontaminate the surface of the suit before the worker
exits the area. The exhaust air from
the suit area is filtered by two sets of high efficiency particulate air/HEPA
filters installed in series. A
duplicate filtration unit, exhaust fan, and an automatically starting emergency
power source are provided. The air
pressure within the suit area is greater than that of any adjacent area. Emergency lighting and communication systems
are provided. All penetrations into the
internal shell of the suit are sealed.
A double-doored autoclave is provided for decontaminating waste
materials to be removed from the suit areas.
Appendix G - Table 1. Possible Alternate Combinations Of Physical And Biological Containment Safeguards
Classification
of Physical & Biological Containment |
Alternate
Physical Containment Laboratory Laboratory Laboratory Facilities Practices Equipment |
Alternate Biological
Containment |
BL3/HV2 BL3/HV1 BL4/HV1 |
BL3 BL3 BL3 BL3 BL3 BL4 BL3 BL3 BL3 BL3 BL3 BL2 BL4 BL4 BL4 BL4 BL4 BL3 |
HV2 HV1 HV1 HV2 HV1 HV2 |
BL - Biosafety Level
HV - Host-Vector System
Appendix G-III-A.
Biosafety in Microbiological and Biomedical Laboratories, 4th
edition, May, 1999, DHHS, Public Health Service, Centers for Disease Control and Prevention,
Atlanta, Georgia, and National Institutes of Health, Bethesda, Maryland.
Appendix G-III-B.
Biosafety in Microbiological and Biomedical Laboratories, 3rd
edition, May 1993, U.S. DHHS, Public Health Service, Centers for Disease
Control and Prevention, Atlanta, Georgia, and NIH, Bethesda, Maryland.
Appendix G-III-C.
National Cancer Institute Safety Standards for Research Involving
Oncogenic Viruses, U.S. Department of Health, Education, and Welfare
Publication No. (NIH) 75-790, October 1974.
Appendix G-III-D.
National Institutes of Health Biohazards Safety Guide, U.S.
Department of Health, Education, and Welfare, Public Health Service, NIH, U.S.
Government Printing Office, Stock No. 1740-00383, 1974.
Appendix G-III-E.
A. Hellman, M. N. Oxman, and R. Pollack (eds.), Biohazards in
Biological Research, Cold Spring Harbor Laboratory 1973.
Appendix G-III-F.
N. V. Steere (ed.), Handbook of Laboratory Safety, 2nd edition,
The Chemical Rubber Co., Cleveland, Ohio, 1971.
Appendix G-III-G.
Bodily, J. L, "General Administration of the Laboratory," H.
L. Bodily, E. L. Updyke, and J. O. Mason (eds.), Diagnostic Procedures for
Bacterial, Mycotic, and Parasitic Infections, American Public Health
Association, New York, 1970, pp. 11-28.
Appendix G-III-H.
Darlow, H. M. (1969).
"Safety in the Microbiological Laboratory," in J. R. Norris
and D. W. Robbins (eds.), Methods in Microbiology, Academic Press, Inc.,
New York, pp. 169-204.
Appendix G-III-I.
The Prevention of Laboratory Acquired Infection, C. H. Collins,
E. G. Hartley, and R. Pilsworth, Public Health Laboratory Service, Monograph
Series No. 6, 1974.
Appendix G-III-J.
Chatigny, M. A., "Protection Against Infection in the
Microbiological Laboratory: Devices and
Procedures," in W. W. Umbreit (ed.), Advances in Applied Microbiology,
Academic Press, New York, New York, 1961, 3:131-192.
Appendix G-III-K.
Horsfall, F. L. Jr., and J. H. Baner, Individual Isolation of
Infected Animals in a Single Room, J. Bact., 1940, 40, 569-580.
Appendix G-III-L.
Biological safety cabinets referred to in this section are classified as
Class I, Class II, or Class III cabinets.
A Class I is a ventilated cabinet for personnel protection having an
inward flow of air away from the operator.
The exhaust air from this cabinet is filtered through a high efficiency
particulate air/HEPA filter. This
cabinet is used in three operational modes:
(i) with a full-width open front, (ii) with an installed front closure
panel (having four 6-inch diameter openings) without gloves, and (iii) with an
installed front closure panel equipped with arm-length rubber gloves. The face velocity of the inward flow of air
through the full-width open front is 75 feet per minute or greater. A Class II cabinet is a ventilated cabinet
for personnel and product protection having an open front with inward air flow
for personnel protection, and HEPA filtered mass recirculated air flow for
product protection. The cabinet exhaust
air is filtered through a HEPA filter.
The face velocity of the inward flow of air through the full-width open
front is 75 feet per minute or greater.
Design and performance specifications for Class II cabinets have been
adopted by the National Sanitation Foundation, Ann Arbor, Michigan. A Class III cabinet is a closed-front
ventilated cabinet of gas tight construction which provides the highest level
of personnel protection of all biosafety safety cabinets. The interior of the cabinet is protected
from contaminants exterior to the cabinet.
The cabinet is fitted with arm-length rubber gloves and is operated
under a negative pressure of at least 0.5 inches water gauge. All supply air is filtered through HEPA
filters. Exhaust air is filtered
through two HEPA filters or one HEPA filter and incinerator before being discharged
to the outside environment. National
Sanitation Foundation Standard 49. 1976.
Class II (Laminar Flow) Biohazard Cabinetry, Ann Arbor, Michigan.
Appendix G-III-M.
Biosafety Level 1 is suitable for work involving agents of unknown or
minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the
general traffic patterns in the building.
Work is generally conducted on open bench tops. Special containment equipment is not required
or generally used. Laboratory personnel
have specific training in the procedures conducted in the laboratory and are
supervised by a scientist with general training in microbiology or a related
science (see Appendix G-III-B, Footnotes
and References of Appendix G).
Appendix G-III-N.
Biosafety Level 2 is similar to Level 1 and is suitable for work
involving agents of moderate potential hazard to personnel and the
environment. It differs 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; and (3) certain procedures in which infectious aerosols are created
are conducted in biological safety cabinets or other physical containment
equipment (see Appendix G-III-B, Footnotes
and References of Appendix G).
Appendix G-III-O.
Office of Research Safety, National Cancer Institute, and the Special
Committee of Safety and Health Experts, Laboratory Safety Monograph: A Supplement to the NIH Guidelines for
Recombinant DNA Research, NIH, Bethesda, Maryland 1978.
Appendix G-III-P.
Biosafety Level 3 is applicable to clinical, diagnostic, teaching,
research, or production facilities in which work is conducted 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 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 protective 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 BL3 (e.g., access zone, sealed
penetrations, and directional airflow, etc.).
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 where facility features satisfy BL2 recommendations provided the
recommended "Standard Microbiological Practices," "Special
Practices," and "Containment Equipment" for BL3 are rigorously
followed. The decision to implement
this modification of BL3 recommendations should be made only by the Principal
Investigator.