APPENDIX D— 29 
normal cell cultures or with susceptible ani- 
mals, especially the normal mouse colony 
(25). 
19. The surgeon’s mask o^ gauze or filter 
paper Is of little value for personal respira- 
tory protection (29). It Is designed to pre- 
vent escape of droplets from the nose or 
mouth (23G). If biohazards demand respira- 
tory protection, then nothing but a full face 
respirator or ventilated hood will suffice. A 
half-mask respirator does not protect the 
eyes, which are an unevaluated avenue of in- 
fection through the, conjunctiva and the 
nasolacrimal duct (5, 8) . 
20. Nonspecific contamination by environ- 
mental organisms from humans, animals, 
equipment, containers for specimens or sup- 
plies, and outside air is a complication that 
may affect or Invalidate the results of an 
experiment. The human sources of this con- 
tamination are evaluated as follows : 
(i) Sneezing, coughing and talking (23A, 
24A). Sneezing, variously reported to gen- 
erate as many 32,000 or 1,000,000 droplets be- 
low 100 microns In diameter; coughing, which 
produces fewer and larger droplets; and 
talking, which has, been reported to average 
only 250 droplets when speaking 100 words, 
show great differences between persons In 
regard to the number of microorganisms 
aerosolized. As a general rule. It may be said 
that these actions by normal healthy per- 
sons may play a less Important role in trans- 
mission or airborne Infection to humans or 
experimental materials than does liberation 
of microorganisms from human skin. 
(II) Dispersal of bacteria from human skin. 
There Is a tremendous variation in the num- 
ber of bacteria shed from the skin by a 
clothed subject. For instance, in one study, 
the number varied from 6,000 to 60,000 per 
minute (23C). These bacteria were released 
on skin scales which were of a size that 
could penetrate the coarse fabric used for the 
laboratory and surgical clothing hi the test 
(23D) . Dispersal of skin bacteria was several 
times greater from below the waist than from 
upper parts of the body (24D). Effective re- 
duction Is accomplished by use of closely- 
woven or impervious clothing fitted tightly 
at the neck, wrists, and ankles to prevent the 
clothing from acting as a bellows that dis- 
perses air carrying skhi scales laden with 
bacteria (23B) . Such clothing sometimes 
Is too warm to work In. It was found that a 
significant reduction in dispersal of bacteria 
occurred with the wearing of close-fitting 
and closely-woven underpants beneath the 
usual laboratory clothing (23D) . The purpose 
of this summary is to alert laboratory per- 
sonnel to the existence of this source of con- 
tamination (9). 
(III) Prolific dispersal of bacteria occurs 
from Infected abrasions, small pustles, bolls, 
and skin disease (23P, 24B). Washing the le- 
sions with germicidal soap will greatly de- 
crease the number of organisms on the skin 
and dispersal Into the air. Healthy nasal car- 
riers who generate aerosolized staphylococ- 
ci usually can be Identified by the presence 
of heavy contamination of their fingers, face, 
and hair (23E). This point may be useful 
In investigating the source of staphylococcal 
contamination of cell lines. 
(Iv) Footwear. In moderate and high risk 
situations, shoes reserved for only laboratory 
use have been recommended as a precaution 
against transp>ortlng spilled infectious agents 
outside the laboratory. However, In experi- 
ments during which reduction of potential 
contamination of experimental materials Is 
Important, laboratory-only shoes can reduce 
the microbial load brought into the labora- 
tory each day by street shoes. Shoes are ef- 
ficient transporters. In one study, there were 
4 to 850 times as many bacteria per square 
centimeter on the laboratory footwear as on 
the fioor Itself (3<H. 
V. CARE AND USE OF LABORATORY ANIMALS 
(10,32-37) 
A. Care and Handling 
1. Special attention must be given to the 
humane treatment of all laboratory animals 
in accordance with the Animal Welfare Act 
of 1970. The Implementing rules and regula- 
tions appear in the Code of Federal Regula- 
tions (CFR) Title 9, Chapter 1, Subchapter 
A, parts 1, 2. 3. Recommended provisions and 
practices that meet the requirements of the 
Act have been published by the U.S. Public 
Health Service (32). 
2. There are specific minimum require- 
ments (33) concerning the caging, feeding, 
watering, and sanitation for dogs, cats, 
guinea pigs, hamsters, rabbits, and non- 
humap primates. To meet these require- 
ments, the animal room supervisor must 
have a copy of 9 CFR Chapter 1. Subchapter 
A, Parts 1, 2, 3. 
3. Each laboratory should establish pro- 
cedures to ensure the use of animals that 
are free of disease prejudicial to the proposed 
experiments and free from carriers of dis- 
ease or vectors, such as ectoparasites, which, 
endanger other experimental animals or per- 
sonnel (10). 
B. Cages Housing Infected Animals (10) 
1. Careful handling procedures should be 
employed to minimize the dissemination of 
dust from cage refuse and animals. 
2. Cages should be sterilized by autoclav- 
ing. Refuse, bowls and watering devices 
should remain in the cage during steriliza- 
tion. 
3. All watering devices should be of the 
"non-drip” type. 
4. Cages should be examined each morning 
and at each feeding time so that dead ani- 
mals can be removed. 
5. Heavy gloves should be worn when feed- 
ing, watering, handling, or removing infected 
animals. Bare hands should NEVER be placed 
in the cage to move any object therein. 
6. When animals are to be injected with 
biohazardous material, the animal caretaker 
should wear protective gloves and the labora- 
tory workers should wear surgeons gloves. 
Animals should be properly restrained to 
avoid accidents that might result In dissemi- 
nating biohazardous material, as well as to 
prevent injury to the animal and to person- 
nel. 
7. Animals exposed' to biohazardous aero- 
sols should be housed In ventilated cages. In 
gas-tight cabinet systems, or In rooms de- 
signed for protection of personnel by use of 
ventilated suits. 
8. Animals inoculated by means other than 
by aerosols should be housed in equipment 
suitable for the level of risk involved. 
9. Infected animals to be transferred be- 
tween buildings should be placed In venti- 
lated cages or other aerosol-proof containers. 
10. The oversize canine teeth of large mon- 
keys present a particular biting hazard; these 
are important in the potential transmission 
of naturally-occurring, and very dangerous, 
monkey virus Infections. Such teeth should 
be blunted or surgically removed by a veteri- 
narian. 
11. Presently available epidemiological evi- 
dence Indicates that Infectious hepatitis may 
be transmitted from nonhuman primates 
(typically chimpanzees) to man. Newly Im- 
ported animals may be naturally Infected 
with this disease, and persons In close con- 
tact with such animals may become Infected. 
After six months residence In this country, 
chimpanzees apparently no longer transmit 
the disease. A record should be' maintained 
for each newly Imported animal. A sign 
should be posted at rooms housing these ani- 
mals to warn that the animals are poten- 
tially Infectious. 
C. General Guidelines that Apply to Animal 
Room Maintenance (10) 
1. Doors to animal rooms should be kept 
closed at all times except for necessary en- 
trance and exit. 
2. Unauthorized persons should not be per- 
mitted to enter animal rooms. 
3. A container of disinfectant should be 
kept In each animal room for disinfecting 
gloves and hands, and for general decontam- 
ination, even though no infectious animals 
are present. Hands, floors, walls, and cage 
racks should be washed with an approved 
disinfectant at the recommended streng^th as 
frequently as the supervisor directs. 
4. Floor drains In animal rooms, as well 
as floor drains throughout the building 
should be flooded with water or dlninfectant 
periodically to prevent backup of sewer gases. 
5. Shavings or other refuse on floors 
should not be washed down the floor drain 
because such refuse clogs the sewer lines. 
6. An Insect and rodent control program 
should be maintained In all animal rooms 
and in animal food storage areas. 
7. Special care should be taken to prevent 
live animals, especially mice, from finding 
their way Into disposable trash. 
D. Necropsy Rules for Infected Animals (10) 
1. Necropsy of Infected animals should be 
carried out by trained personnel In Biological 
Safety Cabinets with the binged glass panel 
down. The glove port panel with or without 
attached gloves, and a respirator should be 
used at the discretion of the supervisor. 
2. Surgeons gowns should be worn over 
laboratory clothing during necropsies. 
3. Rubber gloves should be worn when 
performing necropsies. 
4. The fur of the animal should be wetted 
with a suitable disinfectant. 
5. Small animals should be pinned down 
or fastened on wood or metal In a metal tray. 
6. Upon completion of necropsy, all po- 
tentially biohazardous material should be 
placed In suitable containers and sterilized 
immediately. 
7. Contaminated instruments should be 
placed in a horizontal bath containing a suit- 
able disinfectant. 
8. The Inside of the Biological Safety Cab- 
inets and other potentially contaminated 
surfaces should be disinfected with a suit- 
able germicide. 
9. Grossly contaminated rubber gloves 
should be cleaned in disinfectant before re- 
moval from the hands, preparatory to sterili- 
zation. 
10. Dead animals should be placed In 
proper leak-proof containers, autoclaved and 
properly tagged before being placed outside 
for removal and Incineration. 
VI. DECONTAMINATION AND DISPOSAL 
( 7 , 10 , 38 - 42 ) 
A. Introduction 
Available date on the efficacy of various 
decontaminants for etlologlc agents Indicate 
that no major surprises will be forthcoming 
regarding the susceptibility of organisms 
containing recombinant DNA molecules. In 
the absence of adequate Information, tests to 
determine the efficacy of candidate decon- 
taminants should be conducted with the 
specific agent of interest. The goal of de- 
contamination Is not only the protection of 
personnel and the environment from ex- 
posure to Infectious agents, but also the 
prevention of contamination of experimental 
materials by a variable, persistent, and un- 
wanted background of microorganisms. This 
additional factor should be considered In 
selecting decontamination materials and 
methods. 
