APPENDIX D— 12 
P2 Level (Low) . A laboratory suitable 
for experiments involving recombinant 
DNA molecules requiring physical con- 
tainment at the P2 level Is similar in 
construction and design to the PI labora- 
tory. The P2 laboratory must have access 
to an autoclave within the building; it 
may have a Biological Safety Cabinet."* 
Work which does not produce a consider- 
able aerosol is conducted on the open 
bench. Although this laboratory is not 
separated from the general traffic pat- 
terns of the building, access to the labo- 
ratory is limited when experiments re- 
quiring P2 level physical containment 
are being conducted. Experiments of 
lesser biohazard potential can be carried 
out concurrently in carefully demarcated 
areas of the same laboratory. 
The P2 laboratory is commonly used 
for experiments involving microorga- 
nisms of low biohazard such as those 
which have been classified by the Center 
for Disease Control as Class 2 agents (5) . 
The following practices shall apply to 
all experiments requiring P2 level physi- 
cal containment: <i) Laboratory doors 
shall be kept closed while experiments 
are in progress, (ii) Only persons who 
have been advised of the potential bio- 
hazard shall enter the laboratory, (iii) 
Children under 12 years of age shall not 
enter the laboratory, (iv) Work surfaces 
shall be decontaminated daily and im- 
mediately following spills of recombi- 
nant DNA materials, (v) Liquid wastes of 
recombinant DNA materials shall be de- 
contaminated before disposal, (vi) Solid 
wastes contaminated with recombinant 
DNA materials shall be decontaminated 
or packaged in a durable leak-proof con- 
tainer before removal from the labora- 
tory. Packaged materials shall be dis- 
posed of by incineration or sterilized be- 
fore disposal by other methods. Contam- 
inated materials that are to be processed 
and reused (l.e., glassware) shall be de- 
contaminated before removal from the 
laboratory, (vii) Pipetting by mouth is 
prohibited; mechanical pipetting devices 
shall be used, (viii) Eating, drinking, 
smoking, and storage of food are not 
permitted in the working area, (lx) Fa- 
cilities to wash hands shall be available 
within the laboratory. Persons handling 
recombinant DNA materials should be 
encouraged to wash their hands fre- 
quently and when they leave the labora- 
tory. (x) An Insect and rodent control 
program shall be provided, (xi) The use 
of laboratory gowns, coats, or uniforms 
Is required. Such clothing shall not be 
worn to the lunch room or outside the 
building, (xii) Animals not related to the 
experiment shall no* be permitted in the 
laboratory, (xiii) Biological Safety Cabi- 
nets ' and/or other physical containment 
equipment shall be used to minimize the 
hazard of aerosolizatlon of recombinant 
DNA materials from operations or de- 
vices that produce a considerable aerosol 
(e.g., blender, lyophillzer, sonlcator, 
shaking machine, etc.) . (xlv) Use of the 
hypodermic needle and syringe shall be 
avoided when alternate methods are 
available. 
’ Footnotes at end of article. 
P3 Level (Moderate). A laboratory 
suitable for experiments involving re- 
combinant DNA molecules requiring 
physical containment at the P3 level has 
special engineering design features and 
physical containment equipment. The 
laboratory is separated from areas which 
are open to the general public. Separa- 
tion is generally achieved by controlled 
access corridors, air locks, locker rooms 
or other double-doored facilities which 
are not available for use by the general 
public. Access to the laboratory is con- 
trolled. Biological Safety Cabinets^ are 
available within the controlled laboratory 
area. An autoclave shall, be available 
within the building and preferably with- 
in the controlled laboratory area. The 
surfaces of walls, floors, bench tops, and 
ceilings are easily cleanable to facilitate 
housekeeping and space decontamina- 
tion. 
Directional air flow is provided within 
the controlled laboratory area. The ven- 
tilation system is balanced to provide for 
an inflow of supply air from the access 
corridor into the laboratory. The gen- 
eral exhaust air from the laboratory is 
discharged outdoors and so dispersed to 
the atmosphere as to prevent reentry 
into the building. No recirculation of 
the exhaust air shall be permitted with- 
out appropriate treatment. 
No work in open vessels involving hosts 
or vectors containing recombinant DNA 
molecules requiring P3 physical contain- 
ment is conducted on the open bench. 
All such procedures are confined to Bio- 
logical Safety Cabinets.* 
The following practices shall apply to 
aU experiments requiring P3 level physi- 
cal containment: (i) The universal bio- 
hazard sign is required on all laboratory 
access doors. Only persons whose entry 
into the laboratory is required on the 
basis of program or support needs shall 
be authorized to enter. Such persons shall 
be advised of the potential biohazards be- 
fore entry and they shall comply with 
posted entry and exit procedures. Chil- 
dren under 12 years of age shall not enter 
the laboratory, (ii) Laboratory doors 
shall be kept closed while experiments 
are in progress, (iii) Biological Safety 
Cabinets * and other physical contain- 
ment equipment shall be used for all 
procedures that produce aerosols of re- 
combinant DNA materials (e.g., pipetting, 
plating, flaming, transfer operations, 
grinding, blending, drying, sonicating, 
shaking, etc.), (iv) The work surfaces of 
Biological Safety Cabinets* and other 
equipment shall be decontaminated fol- 
lowing the completion of the experi- 
mental activity contained within them, 
(v) Liquid wastes containing recombi- 
nant DNA materials shall be decontami- 
nated before disposal. Solid wastes con- 
taminated with recombinant DNA ma- 
terials shall be decontaminated or pack- 
aged in a durable leak-proof container 
before removal from the laboratory. 
Packaged material shall be sterilized be- 
fore disposal. Contaminated materials 
that are to be processed and reused (l.e., 
glassware) shall be sterilized in the con- 
trolled laboratory area or placed in a dur- 
able leak-proof container before removal 
from the controlled laboratory area. This 
container shall be sterilized before the 
materials are processed, (vll) Pipetting 
by mouth is prohibited; mechanical pi- 
petting devices shall be used^ (vll) Eating 
drinking, smoking, and storage of food 
are not permitted in the laboratory, (ix) 
Facilities to wash hands shall be availa- 
ble within the laboratory. Persons shall 
wash hands after experiments Involving 
recombinant DNA materials and before 
leaving the laboratory, (x) In insect hnd 
rodent control program shall be provided, 
(xi) Laboratory clothing that protects 
street clothing (i.e., long sleeve solid- 
front or wrap-arotmd gowns, no-button 
or slipover jackets, etc.) shall be worn in 
the laboratory. FRONT-BUTTON LAB- 
ORATORY COATS ARE UNSUITABLE. 
Gloves shall be worn when handling re- 
combinant DNA materials. Provision for 
laboratory shoes is recommended. Labo- 
ratory clothing shall not be worn out- 
side the laboratory and shall be decon- 
taminated before it is sent to the laun- 
dry. (xii) Raincoats, overcoats, topcoats, 
coats, hats, caps, and such street outer- 
wear shall not be kept in the laboratory. 
(xiii) Animals and plants not related 
to the experiment shall not be permitted 
in the laboratory, (xiv) Vacuum lines 
shall be protected by filters and liquid 
traps. (XV) Use of the hypodermic needle 
and syringe shall be avoided when alter- 
nate methods are available, (xvi) If ex- 
periments of lesser biohazard potential 
are to be conducted in the same labora- 
tory concurrently with experiments re- 
quiring P3 level physical containment 
they shall be conducted only in accord- 
ance with all P3 level requirements, 
(xvii) Experiments requiring P3 level 
physical containment can be conducted 
in laboratories where the directional air 
flow and general exhaust air conditions 
described above cannot be achieved, pro- 
vided that this work is conducted in ac- 
cordance with all other requirements 
listed and is contained in a Biological 
Safety Cabinet * with attached glove 
ports and gloves. All materials before re- 
moval from the Biological Safety Cabi- 
net * shall be sterilized or transferred to a 
non-breakable, sealed container, which 
is then removed from the cabinet through 
a chemical decontamination tank, auto- 
clave, ultraviolet air lock, or after the 
entire cabinet has been decontaminated. 
P4 Level (High). Experiments involv- 
ing recombinant DNA molecules requir- 
ing physical containment at the P4 level 
shall be confined to work areas in a facil- 
ity of the type designed to contain micro- 
organisms that are extremely hazardous 
to man or may cause serious epidemic 
disease. The facility is either a separate 
building or it is a controlled area, within 
a building, which is completely isolated 
from all oUier areas of tlie building. Ac- 
cess to the facility is imder strict control. 
A specific facility operations manual is 
available. Class m Biological Safety 
Cabinets * are available within work areas 
of the facility. 
A P4 facility has engineering features 
which are designed to prevent the escape 
of microorganisms to the «ivironment 
(14, 15, 16, 17). These features Include: 
