APPENDIX D— 26 
open front with I nwar d air flow for personnel 
protection, and HEPA-flltered recirculated 
mass air flow for product protection. The 
cabinet exhaust air Is Altered through a 
HEPA filter. Two models of this cabinet are 
available. Type 1 and Type 2. 
(I) Type 1. The Type 1 reclrculatps ap- 
proximately 70% of the air. The exhaust 
air from this cabinet may discharge into the 
laboratory or be diverted out of the labora- 
tory. This cabinet is suitable for CDC classes 
of etlologlc agents 1, 2, and 3. Vapors or 
gases which are hazardous from a toxic, 
radioactive, or flammability standpoint 
should not be used in this cabinet because of 
the high quantity of recirculated air. 
(II) Type 2. The Type 2 cabinet recircu- 
lates approximately 30% of the air. The ex- 
haust air from this cabinet is normally 
ducted out of the laboratory through a 
HEPA filter and, occasionally, an activated 
charcoal filter depending on the operation. 
The cabinet may be used with gases or 
vapors that are hazardous from a toxic, 
radioactive, or flammability standpoint. How- 
ever, any consideration of use of such ma- 
terials should be evaluated carefully from 
the standpoint of bulld-up to dangerous 
levels and problems of decontamination of 
the cabinet. See Table I fbr ventilation re- 
quirements, agent use limitations, and min- 
imum perforniance requirements. 
3. Class III. A closed front ventilated cabi- 
net of gas-tight construction providing total 
protection for personnel and product from 
contaminants exterior to the cabinet. The 
cabinet Is operated under a negative pressure 
of at least 0.5 Inches water gauge. All supply 
air Is HEPA-filtered. Exhaust air Is HEPA- 
filtered or Incinerated to protect the envi- 
ronment. This cabinet, fitted with arm length 
rubber gloves, provides the highest contain- 
ment of these three classes of cabinets and 
Is utilized for all activities Involving high 
risk agents (l.e., CDC etlologlc agents, class 
4) . See Table I for ventilation requirements, 
agent use limitations, and minimum per- 
formance requirements. 
The Integrity of any cabinet depends on 
initial and periodic evaluation to meet es- 
tablished performance tests. Table I outlines 
the minimum performance required to as- 
sure that the cabinets will provide protec- 
tion of personnel and the environment. 
Table 1— Biological safety cahinets, safety performance requirements, and specifica- 
tions, June 1976 
Performance requirements 
Cabinet 
Use classification 
Face velocity 
Exhaust air (cubic 
feet per minute) 
Leak tightness 
filter 
efficiency 
(percent) 
DNA> 
CDC • 
(linear feet 
per minute) 
4-ft hood 
6-ft hood 
Class I. 
P1-P3 
1-3 
75 
200 
300 
Not applicable 
99.97 
Class II, type 1 
P1-P3 
1-3 
75 
260 
400 
Oas tight; leak cate 
<1 by 10-* cm*^ 
2-in water gage 
pressure. 
99.97 
Class II, type 2 
P1-P3 
1-3 
100 
250 
360 
Pressure tight ; no air/ 
soap bubble at 2-in 
water gage pressure. 
Gas tight; leak rate 
<1 by 10-« cm>/s at 
3-in water gage 
pressure. 
99.97 
Class in 
P4 
4 
(>) 
(<) 
(*) 
99.97 
• For work with recombinant DNA molecules. 
> Center tor Disease Control (U.S. Public Health Service). 
> Not applicable. 
‘ Based on 1 vol. of air change each 3 min. In the absence of unusual heat or moisture that would require more air 
changes. 
n. UNIVERSAL BIOHAZARD WARNING SYMBOL (1) 
The biological hazard warning symbol 
(biohazard symbol) specified herein shall be 
used to signify the actual or pK>tentlal pres- 
ence of a biohazard and to Identify equip- 
ment, containers, rooms, materials, experi- 
mental animals or combinations thereof 
which contain or are contaminated with 
viable hazardous agents. 
The blohaaard symbol shall bo designed 
and proportioned as Illustrated here; 
The symbol shall be as prominent as prac- 
tical, and of a size consistent with the size 
of the equipment or material to which it is 
afidxed, provided the proportions shown above 
are maintained, and, in any case, that the 
symbol can be easily seen from as many 
directions as possible. 
Except when circumstances do not permit, 
the symbol shall b^ oriented with one of the 
three open circles pointed up and the other 
two forming a base. 
The symbol color shall be a fluorescent 
orange or orange-red color.* Background 
color is optional as long as there is sufiaclent- 
contrast for the symbol to be clearly defined. 
BIOHAZAR 
>••1 Ct*^ VlN Oraac* of th« Switaac Brother*, !*&. 1* elt*4 a* an axaapla, 
a»C «a mBor*«Mac. 
The biohazard symbol shall be used or dis- 
played only to signify the actual or potential 
presenc^f biological hazard. 
Appropriate wording may be used in asso- 
ciation with the symbol to Indicate the na- 
ture or Identity of the hazard, name of indi- 
vidual responsible for its control, precau- 
tionary Information, etc., but never should 
this Information be superimposed on the 
symbol. 
ADMinANa TO AUTHORUED PEISOIIEL ORIT 
HoMrd id«nKry; . 
ItMponiibl* lnv«fl*gotef; 
tn COM of omorgoncy eofl; 
Doytimo phono Homo phono ^ 
A^orizotion for ontronco moil bo obfoiood from 
tho Itofponiiblo InvosHootor no mod obovo. 
III. LABORATORY TECHNIQUES FOB BIOHAZARD 
CONTROL 
A. Pipetting 
1. No Infectious or toxic materials should 
be fdpetted by mouth ( 2 , 3, 4) . 
2. No infectious mixtures should bo pre- 
pared by bubbling expiratory air through a 
liquid with a pipette (2, 3, 4) . 
3. No infectious material should be blown 
out of pipettes (2, 3, 4) . 
4. Pipettes used for the pipetting of infec- 
tious or toxic materials should be plugged 
with cotton (2, 3, 4) . 
5. Contaminated pipettes should be placed 
horizontally In a pan containing enough 
suitable disinfectant to allow complete im- 
mersion (2, 4, 4) . They should not be placed 
vertically In a cylinder. 
6. The pan and pipettes should be auto- 
claved as a unit and replaced by a clean pan 
with fresh disinfectant (2, 3, 4) . 
7. Infectious material should not be mixed 
by alternate suction and expulsion through 
a pipette (2, 3, 4) . 
8. Mark-to-mark pipettes are preferable to 
other types, as they do not require expulsion 
of the last drop (6) . 
9. Discharge should be as close as possible 
to the fluid or agar level, or the contents 
should be allowed to run down the waU of 
the tube or bottle whenever possible — ^not 
dropped from a height (6) . 
10. A disinfectant-wetted towel over the 
immediate work surface is useful In some 
cases to minimize the splash from accidental 
droppage (9) . 
B. Syringes and Needles (9) 
1. To lessen the chance of accidental In- 
jection, aerosol production or spills, avoid 
unnecessary use of the syringe and needle. 
For Instance: 
(I) Use the needle for parenteral injections 
but use a blimt needle or a cannula on the 
syringe for oral or Intranasal inoculations. 
(II) Do not use a syringe and needle as a 
substitute for a pipette In making dilutions 
of dangerous fluids. 
2. Use the syringe and needle in a Biologi- 
cal Safety Cabinet only and avoid quick and 
unnecessary movements of the hand holding 
the syringe. 
3. Examine glass syringes for chips and 
cracks, and needles for barbs and plugs. 
