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APPLIED MICROBIOLOGY, May 1968, p. 762-771 Vol. 16, No. 5
Copyright © 1968 American Society for Microbiology Printed il U.S.A.
New Ventilated Isolation Cage
REGINALD 0. COOK
Nationial Environimenital Health Sciences Center, Research Trianigle Park, Northl Carolinia 27709
Received for publication 16 February 1968
A multifunction lid has been developed for a commercially available transparent
animal cage which permits feeding, watering, viewing, long-term holding, and local
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transport of laboratory rodents on experiment while isolating the surrounding en-
vironment. The cage is airtight except for its inlet and exhaust high-efficiency
particulate air filters, and it is completely steam-sterilizable. Opening of the cage's
feed and water ports causes an inrush of high velocity air which prevents back-migra-
tion of aerosols and permits feeding and watering while eliminating need for chem-
ical vapor decontamination. Ventilation system design permits the holding in ad-
jacent cages of animals infected with different organisms without danger of cross-
contamination; leaves the animal room odor-free; reduces required bedding changes
to twice a month or less, and provides investigators with capability to control
precisely individual cage ventilation rates. Forty-eight cages can be conveniently
placed on a standard NIH "shoebox" cage rack (60 inches wide X 28 inches deep X
74 inches high) fitted with a simple manifold exhaust system. The entire system is
mobile, requiring only an electrical power outlet. Principal application of the caging
system is in the area of preventing exposure of animal caretakers to pathogenic
substances associated with the animal host, and in reducing handling of animals
and their exposure to extraneous contamination.
The holding of laboratory animals infected sterilizable entry lock, and the actual process of
with organisms known to be transmissible to man feeding and watering is done through gloves which
has long been recognized as a problem, both in are part of the barrier. Isolation systems of this
preventing infection of those caring for the type for germ-free animals were reported by
animals and in preventing dissemination of the Reyniers et al. (rigid stainless steel) in 1943 and
organisms to healthy control animals housed 1959 (14, 15) and by Trexler et al. (flexible
nearby (2, 4, 7, 9, 11, 16). More recently, organ- plastic) in 1957 (19). Phillips et al. (10) reported
nisms thought to be species-specific have been a flexible plastic version for infectious work in
shown to cross species barriers when applied in 1955. The gas-tight stainless-steel Biological
the highly concentrated preparations now avail- Safety Cabinets (4, 22) pioneered by the U.S.
able to virologists (1, 17, 18). Subsequent recogni- Army Biological Laboratories at Fort Detrick,
tion of the potential hazards associated with the Md., for highly infectious work represent the
investigation of high potency, wide host range present ultimate in this approach to isolation, but
oncogenic or leukemogenic viruses (Hellman, their use for holding animals on experiment is
unpublished data) led to increased interest in the economically out of reach for most laboratories.
development and use of containment facilities Basic drawbacks associated with the large (2 ft
(12, 13). As a part of the overall containment X 4 ft X 2 ft) negative pressure flexible plastic
problem, the need was recognized for a compact, isolators in use at the National Institutes of Health
simple, economical, effective rodent isolation were the floor space required (about 48 ft2) for
cage for routine infectious or potentially infectious the number of animals held (about 12 small
work. This paper describes the development of a shoebox cages), the transfer lock sterilizations,
ventilated isolation cage system to meet this need. which must be carried out each time food, bed-
There are two methods by which an isolation ding, water, or animals are passed in or out, and
system for small animals can be constructed and potential for airborne cross-contamination within
maintained. One approach is to construct a the enclosure. The transfer lock decontamination
comparatively large isolated area and hold the was by a chemical vapor sterilant, an inherently
animals in open individual cages inside. In this messy process requiring several hours.
system, cages, food, bedding, and other essentials The alternative approach to the biohazards
are passed into the isolation chamber through a isolation problem is to make each individual cage
762'_t
VOL. 16, ] 968 NEW ISOLATION CAGE 763
the isolation unit-self-contained and inde- Plastics Inc., Federalsburg, Md.) and a gasketing
pendent in and of itself. and clamping arrangement to seal them together.
The development of such a caging system was The resulting cage unit permits the holding,
undertaken as part of the biohazards control transporting, viewing, feeding, and watering of
program of the Environmental Services Branch, the animals, while containing biological aerosols
National Institutes of Health in the belief that generated by them and drastically reducing
once the technical problems of air filtration, potential for entry of extraneous contamination
containment, and food and water introduction from outside.
were solved, a system consisting of many self- Figures 1 and 2 show, respectively, an "ex-
contained, fully independent isolation cages ploded" view of the cage, and a view of the cage
would be superior to the large isolator-open cage rack and ventilation system with cages connected.
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system in many instances. In Fig. 1, air enters the cage through the supply
The system developed as a result of this effort filter (11) and leaves through the exhaust filter
has been fabricated and is now in use (in the (10). Food, and in special cases animals, enter
laboratory of S. E. Stewart of the National Cancer the cage through the feed port (6). Water is
Institute). The nucleus of the isolation system is introduced through the water port (20) into the
a mechanically ventilated isolation cage unit water pan (8). Animals drink at the automatic
whose basic components are a multifunction water valve (18). The venturi orifice (15) regu-
stainless-steel lid, a commercially available lates ventilation air flow.
transparent plastic shoebox cage (from Maryland The exhaust hose (14) conducts filtered air from
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G.3.
H i at ama
FIG. 3. Hamster drinking at automatic water valve.
cages for separation of one cage environment
from another and for independence and mobility
of the complete system.
Following is a discussion relating why, how,
and to what extent each of these requirements
was incorporated in the design now in use.
FIG. 2. Cage rack and ventilation system with cages (i) Visibility. The plastic "shoebox" bottom
connected. (through which the animals can be seen) is
fabricated from polycarbonate resin whose light
the cage to the exhaust manifold attached to the transmissibility is approximately 85% (from
cage rack. Figure 3 shows a hamster drinking at Polycarbonate Resin Handbook, General Electric
the automatic water valve. Figure 4 is an occu- Co.). Figure 5, a photograph taken of a hamster
pant's eye view of the cage lid. inside the cage, is a good illustration of the
EXPERIMENTAL visibility possible. In addition, the exhaust hose
(item 14, Fig. 1) has been made long enough to
The design, modification, and testing of the allow removal of the cage from the cage rack for
ventilated isolation cage was guided by certain close viewing of the occupants while the cage
requirements deemed essential to the acceptance remains connected to the exhaust manifold.
and subsequent successful functioning of any (ii) Air tightness. Air tightness is a critical
compact, independently ventilated isolation requirement of any negative pressure isolation
cage system. These requirements were that the system, which a "biohazard" isolation
individual cage unit should: (i) provide good must be. Air tightness is necessary to maintain system
visibility; (ii) be airtight; (iii) be sterilizable by the negative pressure, to insure no outflow of
steam-autoclaving; (iv) have compact, reliable,
easily obtainable, ultrahigh efficiency inlet and organisms should the source creating the negative
exhaust filters; (v) provide capability for con- pressure fail, to prevent inflow of extraneous
venient local transport while maintaining isola- contamination, and to allow the designer to state
tion; (vi) provide for introduction of feed and with assurance the precise aerodynamic capabil-
water while maintaining isolation without neces- ities and limitations of the system. Manufacturing
sitating chemical vapor decontamination of the specifications established for the ventilation
entry portal; (vii) provide investigators with a isolation cage unit provide for the rejection of any
selection of precisely controlled ventilation rates; cage not airtight against 3 inches water gauge
(viii) be compatible for use with gas-tight Biolog- (w.g.) pressure. Our testing indicates that the
ical Safety Cabinet Systems; (ix) utilize the in- capability of the ventilated isolation cage is much
herent possibilities of a system based on individual greater, with some cages withstanding 1 psi (28.3VOL. 16, 1968 NEW ISOLATION CAGE 765
since it is through these that air, but no con-
taminating or infectious aerosols, must pass. In
biohazards isolation, the exhaust filter is more
critical, since it must contain and collect any
infectious aerosols generated by the animals. The
inlet filter (item 11, Fig. 1) provides contaminant-
free air to the cage in normal operation and pre-
vents backflow of infectious aerosols from within
when the cage is disconnected from the exhaust
manifold or transport. Both filters selected for
use on the ventilated isolation cage are commer-
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cially available as face mask particulate filters.
Both are commercially marketed by the manu-
facturer (Mine Safety Appliances Co., Pittsburgh,
Pa.) with a guarantee of 99.98% retention when
FIG. 4. Occupants eye view ofthe cage lid. challenged by 0.3-,u uniform-diameter dioctyl
phthalate (DOP) smoke in the standard Army
inches w.g.) positive pressure without leaking. Chemical Corps test (20). Retesting of selected
(Positive pressure tests are considered to give a filters by the manufacturer (after as many as 16
surer indication of air tightness than negative live steam autoclavings for 1 hr at 265 F by us)
pressure tests.) Negative pressures as low as gave DOP efficiencies between 99.993 and
minus 12 psi have been maintained in the cage, 99.999% (DeCecco, personal communication).
but it is difficult to prevent leaks at this extremely Harstad et al. (5, 6), after challenging similar
low pressure. Cage negative pressure for the commercially available high-efficiency particulate
mouse-hamster cages now in use is approximately air (HEPA) filters with Ti coliphage particles
1.85 inches w.g. having a number median diameter (NMD) of
(iii) Steam sterilization. To be justifiable eco- 100 m,u, concluded that such filters provided
nomically, an individual cage unit must either be excellent protection against submicron virus
cheap enough to be disposable after one use or particles. Efficiencies of these filters (from four
must be capable of withstanding repeated steam, manufacturers) averaged 99.997%.
chemical vapor, or gas sterilization without In tests conducted by the same investigators on
change in characteristics. Of the many decon- the supply and exhaust filters used on the venti-
taminants for materials that are not heat-labile, lated isolation cage with the 100-m,u NMD Ti
steam autoclaving is almost universally the phage challenge agent, filtration efficiencies
method of choice because it is readily available, proved to be higher than those of the larger com-
clean, and positive. Since development of an mercial filters, even after 16 autoclavings (Har-
effective, disposable cage seemed beyond our stad, personal communication). No attempt was
reach, all components were selected on the basis made to investigate the effect of further autoclav-
of their capability to withstand repeated steam
sterilization.
In evaluation tests, commercially fabricated
(by Ellisco, Inc., Philadelphia, Pa.) ventilated
isolation cage units, excluding filters, have been
autoclaved 65 times (at 265 F for 1 hr) without
decreasing their fitness for service.
An assembled cage, including filters and bed-
ding, can be sterilized in a high-vacuum autoclave
by exposure to steam at 258 F for 15 min after
an autoclave chamber vacuum of 75 mm of Hg
absolute has been reached, thus providing a
sterile cage environment before introduction of
animals. The assembled, sterilized cage can then
be stored indefinitely for later use since the only
air pathway from inside to outside is through the
filters (see next section).
(iv) Filters. It is important to recognize that the
supply and exhaust filters are a part-perhaps FIG. 5. Demonstration of ability to see the inside of
the most important part-of the isolation barrier, the cage.766 COOK APPL. MICROBIOL.
ings upon filter efficiency, because it was pro- (Aerodynamic parameters of the filters are de-
jected that 16 autoclavings represented at least 4 lineated in section vi and vii and in Fig. 6-8.) For
months of use, by which time the filters' resistance the sake of compactness, convenient removal, and
will have increased to a point where they should safety, the filters are screwed into adapters (see
be replaced. Fig. 1) welded into the stainless-steel lid. Thus,
The inlet filter is smaller and less expensive than the filters become a physically strong part of the
the exhaust filter, because it need only filter the barrier, not easily dislodged or broken.
cage ventilation flow of 0.28 ft3 per minute, Highly efficient, compact, consistent, economi-
whereas the exhaust filter must occasionally filter cally justifiable filters are critical to the success of
the much greater (4.2 ft3/min) open feed-port any isolation cage. The filters used on the venti-
containment flow, which bypasses the inlet filter. lated isolation cage represent a major advance
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toward this end. Both are manufactured to mili-
tary specifications on a large scale for use in face
masks, are individually checked for specification
compliance, and are inexpensive enough to be
considered "throwaway" items.
In the biohazards context, it is always prudent
to over-design. Therefore, a comparatively large
(55 ft3/min) filter, equal in manufacturer's guar-
anteed filtering efficiency to the cage filters, is
located atop each cage rack where it refilters all
exhaust air from the cages. Manufacturer's guar-
anteed minimal efficiency then is a minimum of
99.999996%, since filtration by HEPA filters is
accomplished on a matrix basis where the chance
of a particle getting through is based on statistical
probability rather than on its size in relation to a
uniform pore diameter (3).
(vi) Local transport. Once the filters are in place
and the lid is clamped on, the cage can be trans-
ported, since the only air path from inside to out-
side is through the two filters. During transport,
some natural diffusion ventilation does occur
.10 .20
FLOW -
.30
CUBIC FEET PER MINUTE
.40 .50
through the filters because of temperature gradi-
FIG. 6. Cage negative pressure versus ventilation ents. A word of caution, however, is necessary,
rate. Air enters the cage through supplyfilter mechanism because the animals will eventually succumb, ap-
consisting of (J) supplyfilter and holder and (2) threaded parently from buildup of C02, if the cage is not
brass orifice. The curves represent flow through the reconnected to the exhaust manifold for short
supply filter and holder with different orifices attached. intervals. The time span in which the cage may be
Velocity range at
constant manifold
pressure, 2 inch
dimseter port
Velocitie nay deoreaae seightty as filters
load up when ueed to fitter exhauet frarn
negative preaeure "bio-haaarda omg". When
ueed to fitter euppty air for positioo preeeum
(SPF) oagee, design oalte for fitters' protec-
tion by a H.E.P.A. pre-fitter, which will
prevent Load up and inBure no change in port
velocity.
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
PORT VELOCITY - FEET PER MINUTE
FIG. 7. Manifold pressure versus air velocity through open ports in ventilated cage, based on the use offilters
having a resistance of not more than 32 mm nor less than 38 mm water gauge at a flow of 3 ft'/min.VOL. 16, 1968
220
200
180
R160
i40
'120
ml00
= 80
S.~ Rev~rs3. NEW ISOLATION CAGE
Port flow vs. port
velocity. 2 Inc
diamter port
/ fRlter*
/
/
/
/
/Pressure differentil vs.
>
~~~~~~~~~~~~Port
flow vs.
~~~~~~~~~~~~~port
velocity,
, z ~~~~~~~~~~~3-3/8
inch di&-
,, ',, ~~~~~~~~~~~~eter
port.,,
Us
w>Z~~~~~~~~~veocity desired
-hi'
767
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W 60 / _ relating port ftow to port
~~~~~~~~~~~~curve
0
/ /~~~~~~~~4
MoevrticaIZy to inttereectiontof f*ltter ,fZow curve.
Move horisotally to Zeft and read necesaMrmonifoZld pressure.
/ procedure to oad velocity at any give mifold pressure.
20 /~~~~~~~~M 201WAT FIL FLO, CAGEFL P, AN OPEN POTFWAR THE SAA,
.
/ 2WA MANIPOLD PRESSURE IS FILItER DIFFfEMNIAL PRESSSURE l/ITH PORT
~~~~~~~~AND
OPENJ
0.2 0.6 l 0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 7.0
OPEII PORT CAGE AIR FLOW - CUBIC FEET PER NIWIITE
FIG. 8. Ventilated cage rack manifold pressure versus open port cageflow rate and port velocity versus cageflow
rate, based on the use offilters having a resistance ofnot more than 32 mm nor less than 28 mm water gauge at a
flow of3 ft$/min.
safely used without externally produced ventila- was determined to be 2 inches after consultation
tion varies with the number and size of the ani- with animal users and after trials indicated that
mals, so no specific figure can be given. When six anything less would begin to hamper introduction
fully grown mice were placed in a cage, overt of food.
symptoms (ruffled fur) began to appear in about Next, an investigation was made to determine
3 hr, and death occurred in about 8 hr; when only what incoming air velocity would pose an impene-
one fully grown hamster was placed in the cage, trable barrier to airborne particles already within
survival was noted beyond 36 hr. the cage. This air velocity was shown to depend on
(vi) Introduction of food and water. It is best several factors, primarily the shape of the food
that any isolation cage system be designed to mini- port and whether there were sources inside the
mize the number of times a primary barrier (in enclosure imparting a momentum to the aerosols
this cage, the lid) need be dismantled. as they are generated. Therefore, no one answer
In accordance with this concept, a small port applicable to all situations could be determined.
was attached to the lid through which food may However, it was apparent that use of a port shape
be passed into the cage. This port (item 6, Fig. 1) approaching a long narrow cylinder as the inlet
is a diameter cylinder (2 inches high, 2 inches in would ensure aerosol containment at lower veloc-
diameter) having a threaded cap similar to the ities than an unadorned hole in a flat surface.
familiar mason-jar top. When the cap is removed, After containment tests conducted with titan-
food may be simply dropped into the feed hopper ium tetrachloride smoke at inlet air velocities as
inside the cage. A uni-directional stream of air low as 80 ft/min (average fume hood velocity)
flowing from room into cage through the port indicated no escape, an inlet air velocity of 200
during feeding prevents escape of aerosols from ft/min was established for the food ports of initial
within the cage. Such a uni-directional flow of air cages. (Figure 9 shows the air flow pattern at 200
has been shown to be an effective barrier to mi- ft/min.) Design of the cage's ventilation system
crobial agents (8; D.G. Fox, Ph.D. Thesis, Univ. permits the use of velbcities significantly higher or
of Minnesota, Minneapolis, 1967) and is widely lower than 200 ft/min; however, 200 ft/min was
used to exclude contamination in the pharma- judged optimal because it provided a factor of
ceutical and aerospace industry. safety, introduced no turbulence in the bedding,
The following considerations led to the selec- and minimized total quantity of air passing
tion of 2 inches (5 cm) as the port diameter. The through the cage. Undoubtedly, there will be ap-
use of a "moving air" barrier dictated that the plications where other velocities will be preferable.
port be kept as small as possible to minimize the A flow of 4.2 ft3 of air per min is required to
total quantity of air required to produce the de-
sired velocity. However, the port had to be large produce a 200 ft/min air velocity through a 2-inch
enough to allow convenient introduction of both diameter port.
"wet" (potato, carrot, or apple slices) and dry Manufacturer's data and further tests by us
food (pellets). This minimal acceptable diameter indicated that the flow rate-pressure drop charac-768 COOK APPL. MICROBIOL.
cage rack. The animal caretaker then fills each
pan individually by a length of flexible tubing
from the carboy, utilizing gravity flow.
The watering method used in the ventilation
isolation cage is a radical departure from the
traditional water bottle-sipper tube arrangement.
However, it is believed that placing the water
reservoir totally and permanently inside the isola-
tion barrier significantly reduces potential for
escape of infectious aerosols, relative to the poten-
tial for their escape when the water reservoir
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(sipper tube and water bottle) is primarily outside
FIG. 9. Air-flow pattern in the cage with an in7let the barrier and withdrawn for refilling. If a sipper
air velocity of 200 ft/min. tube-water bottle were used, withdrawal of the
sipper tube through the cage top would expose a
teristics of the exhaust filter required the existence contaminated wet surface-a likely source of
of a pressure differential of 2.0 inches w.g. across aerosols. More importantly, the (external) water
the filter to produce a flow of 4.2 ft3/min (see Fig. bottle must be assumed to be full of contaminated
8). Thus, the governing exhaust system aerody- air from inside the cage, since water is made
namic parameter was established, i.e., a negative available to animals from a sipper tube-water
pressure of at least 2.0 inches w.g. must always be bottle by relief (by cage air) of the partial vacuum
maintained just downstream of the exhaust filter above the water. If any water remained in the bot-
for 4.2 ft3/min to flow automatically through an tle, the process of turning the bottle right side up
opened feed port, producing the desired 200 ft/ would result in the water falling to the bottom and
min velocity. the trapped cage air being forced out of the sipper
The 200 ft/min is a minimal "steady state" flow tube in the resulting dynamic turbulence, to say
rate. As the feed port cap is being unscrewed for nothing of the potential for breakage or contact
removal, inward air velocity through the cap with infectious material while the bottle is being
threads and over the lip momentarily reaches handled in preparation for sterilization.
4,000 ft/min (45 mph) before the cage negative Since there is no spillage with the automatic
pressure is dissipated. This momentary high ve- water valve as there is with the sipper tube, a given
locity is significant because it dislodges and car- amount of water lasts longer. In tests conducted
ries away any particles loosely attached to the by us, the 480 ml of water held by the pan and
inside surface of the cap, which might otherwise 200 g of specific pathogen-free rat and mouse diet
be liberated if the cap were accidentally dropped. held by the feed hopper (not shown) was a 10-day
Still, prudence does dictate careful handling of supply for five fully grown Swiss albino mice.
the cap, since its inner surface has been exposed During this 10-day period, the cage was fully self
to air inside the cage. sufficient, i.e., not touched. The 10-day length is
Introduction of water is accomplished in basi- significant, because extraneous contamination
cally the same manner as introduction of food, can only enter a properly fabricated (plans and
and is made possible by the same moving air bar- specifications are available from the Environmental
rier principle. The water pan, which holds about Services Branch, National Institutes of Health,
480 ml (item 8, Fig. 1), is attached to the under- Bethesda, Md.) and set up cage when the feed or
side of the cage lid inside the isolation barrier. water ports, or both, are opened. If absolute con-
Animals drink by pressing the tip of an automatic trol of extraneous contamination is a necessity,
watering valve attached to th,e bottom of the pan. this route can be blocked by carrying out the food
Refilling of the pan takes place through a trans- and water introduction in a downflow "laminar"
parent cylinder extending 2 inches above the lid. air clean bench.
The cylinder is normally capped and made air- (vii) Ventilation. The rate at which ventilation
tight by a laboratory sleeve stopper, which permits air flows through the cage can be conveniently
water to be inserted by syringe if deemed neces- varied from 0 to more than 1.5 ft3/min. However,
sary. Otherwise, the sleeve stopper is removed, for general use, an orifice which sets the flow at
whereupon air at a velocity of 250 ft/min flows approximately 0.28 ft3/min has been selected.
down the cylinder, into the pan, and out into the This flow was arrived at in the following manner.
cage via small holes in the pan's vertical wall. It was first observed that the economics of filter
Water is then poured into the 0.5-inch diameter life and air-handling equipment, as well as con-
cylinder, thereby filling the pan. For efficient re- tamination exclusion, would dictate the lowest
plenishment of water, a carboy is placed atop the possible ventilation rate consistent with the oc-VOL. 16, 1968 NEW ISOLATION CAGE 769
cupants' health and well being. It was initially The desired 0.28 ft3/min cage flow rate could be
postulated that this ventilation rate would fall in produced by a manifold negative pressure (suc-
the range of no more than 10 to 12 air changes tion) as low as 0.35 inches w.g. However, operat-
per hour. (Institute of Laboratory Animal Re- ing manifold pressure could not be set at 0.35
sources, NAS-NRC recommendations call for inches w.g., because it was necessary to maintain
animal rooms to receive a minimum of 10 to 15 2.0 inches w.g. pressure in the manifold to pro-
air changes per hour (21); however, it is doubtful duce the open feed port containment flow of 200
that open cages within such a room individually ft/min (4.2 ft3/min). Therefore, to produce a ven-
receive that many air changes.) Since the venti- tilation flow of 0.28 ft3/min with an applied suc-
lated isolation cage's internal volume is about tion pressure of 2.0 w.g., an energy dissipating
0.28 ft3 (13 inches x 7.5 inches x 5 inches deep), element was added to the inlet filter adapter to
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10 air changes per hour would be given by flow of reduce air flow to the desired level, just as resist-
0.046 ft3/min (1.3 liters per min), a very low flow ance is added to regulate flow of electrons in an
indeed. electrical circuit where voltage is present at a con-
To test this projected ventilation rate, a cage stant level. Since energy in a moving air stream
containing six weanling mice was set up (under can be dissipated by friction and turbulence, many
the supervision of C. D. LeMunyan and C. T. devices could have been used. A venturi-type
Hansen, Animal Production Section, National orifice is often the method of choice where precise
Institutes of Health). Although 0.05 ft3/min was control and long-term accuracy are desired. A
enough to keep the mice healthy, it was noted that standard brand "l 6 inch tube to tube" brass
a flow in the 0.25 to 0.30 ft3/min (7.2 to 8.5 liters refrigeration plumbing elbow which forms a
min) range kept the cage bedding dry also; i.e., venturi-type orifice (see item 15, Fig. 1) was found
urine was being evaporated and carried away as to meet the aerodynamic requirements established
fast as it was voided. The mice were held for 1 for mouse or hamster occupancy. This readily
month in a 0.28 ft3/min flow rate without buildup available elbow is inexpensive, autoclavable, not
of moisture necessitating a change of bedding. In susceptible to chewing damage by animals, and
another test, four females were bred, and their has the aerodynamic property of allowing only
resulting offspring were weaned before odor and small changes in flow when subjected to relatively
accumulation of dry feces necessitated change of large variations in pressure. Because of the latter
bedding. Although this turn of events was not property, it is possible to change the feed port
anticipated, its advantages were immediately containment flow (by increasing or decreasing the
apparent in view of the fact that bedding in a con- manifold pressure) while changing the normal
ventional cage has to be changed approximately flow by only an insignificant amount. For in-
twice a week. Animal caretaker manpower saving stance, with the above ventilation orifice, a con-
would appear to be significant. This feature would tainment velocity of 200 ft/min (4.2 ft3/min) and
also appear advantageous where long-term latent a normal ventilation flow of 0.28 ft3/min is pro-
periods are anticipated after challenge of animals duced by a manifold vacuum of 2 inches w.g.,
and in other circumstances where it is advanta- whereas a 1.5 inch w.g. manifold vacuum pro-
geous to keep handling of animals to a minimum. duces a ventilation flow of 0.24 ft3/min and a feed
A flow rate of 0.28 ft3/min results in a 60 air port containment velocity of 150 ft/min (see
changes per hour ventilation rate (one each min- Fig. 6 and 7).
ute) since the cage's volume is approximately By switching to a different orifice, it is possible
0.28 ft3. At first, this high ventilation rate (by to change the cage ventilation rate while holding
normal standards) might be thought to set up a the manifold pressure and thus the feed port con-
draft, or dehydrate the animals, but neither oc- tainment flow constant (the reverse of the above).
curs. At a 60 air changes per hour ventilation rate, Selection of flows at various points between
average air velocity across the full cross section nearly 0 and 1.50 ft3/min is possible since a num-
area of the cage is only about 1 ft/min. By com- ber of different elbow orifices are available. Thus,
parison, fume hood velocities are generally about while one cage's ventilation rate is 50 air changes
80 to 100 ft/min (approximately 1 mph). Dehy-
dration of the animals does not occur if the incom- per hour, an adjacent cage may be set at 80 air
ing air is at the normal animal room standard of changes per hour, and a third at 120 air changes
45 to 55% relative humidity. The high ventilation per hour. When their respective feed ports are
rate promotes continuous evaporation of the opened, all will be found to experience the same
urine and moisture. Vapor is carried away before containment velocity. Data for several elbow
the relative humidity within the cage has risen orifices whose flow-pressure loss characteristics
significantly above that of the incoming air. have been calibrated by wet test meter are shown770 COOK APPL. MICROBIOL.
in Fig. 6. Costs of these elbow orifices range from Additionally, a source of negative pressure (de-
12 to 26 cents. veloping 2 inches or more w.g. at 4.2 ft3/min flow)
These orifices do produce some noise, as does is made available near the cabinet for connection
any orifice whose function is to accelerate air. to the cage exhaust filter. To transfer animals, the
Air velocity reaches about 3,000 ft/min inside the cage port is slipped under the cabinet port, and
il6 inch tube to tube" orifice at 0.28 ft3/min the two are connected by a slip-fitting sleeve. The
flow, but the elbow is directed toward the cage animals are then dropped from cabinet down the
corner where the horizontal air velocity is quickly then-closed cylinder into the cage. The cabinet
dissipated. The mice and hamsters held in the cage port is normally capped on both ends and is de-
for 1-month periods showed no ill effects. signed to be absolutely airtight, just as the cage
To achieve equal pressures at all exhaust mani- port is, but, if a leak should occur in the connec-
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fold nipples, the pipes and tubing which comprise tion during transfer, the 2 inch w.g. negative
the exhaust system between the cage exhaust filter pressure will draw air from the room into the cage
and exhaust fan must be sized so that energy or negative pressure cabinet at extremely high
losses due to air turbulence do not occur between velocity (4,000 ft/min or more) to prevent escape
these two points. Generally, this means air veloci- of aerosols. For autopsy or for cage changing, the
ties must be kept below 400 ft/min (0.01 inch cage is returned to the biological safety cabinet,
velocity pressure). When the exhaust piping is and the transfer process is repeated.
sized accordingly, manifold pressure (given by a (ix) Cage environment separation and system
permanently installed manometer) can be con- mobility. The ventilated cage can be used on any
verted into feed port velocity or flow rate, or both, cage rack which has been fitted with a proper
by use of Fig. 7 or 8. Cage pressure can be con- manifold. Since each cage is independently con-
verted into cage ventilation by use of Fig. 6 and nected to a separate exhaust manifold nipple by a
cage pressure. Thus, these important flow and slip-fit connection which can be connected and
velocity parameters are always available at a removed by hand, air passing through each cage
glance. is conveyed through the exhaust line, and, having
In spite of the high (60 air changes per hour) passed through the two exhaust filters, into the
ventilation rate per cage, a system composed of building exhaust duct. Thus, cages housing ani-
such cages requires significantly less air than con- mals experimentally exposed to different organ-
ventional animal rooms receiving 15 air changes isms can be held in adjacent cages without air-
per hour, the accepted standard. A 16 ft x 32 ft x borne cross-contamination occurring or animal
10 ft high animal room could accommodate 14 odors escaping into the room.
ventilated isolation cage racks. Not more than The blower and large filter can be mounted on
210 ft3/min (15 ft3/min per rack of 48 cages) the unused top shelf of the five-shelf, 48-cage rack,
would be required to supply 60 air changes per thus making the entire system of 48 ventilated
hour to each cage, whereas 1,280 ft3/min would isolation cages completely mobile. Since an elec-
be required to supply 15 air changes per hour to trical power outlet and proper room air tempera-
an identical room housing conventional open ture are the only external requirements for opera-
cages. Significant ventilation air savings appear tion of the system, it can be set up almost any-
possible where this system can be used. where space is available.
Of paramount importance to the proper aero-
dynamic functioning of the system is the use of an DIscUSSION
exhaust blower with a relatively flat static pres- The maintenance of experimental animals has
sure versus flow curve. In most applications, an stimulated the design of a variety of isolation
exhaust blower delivering a negative pressure of caging and equipment. The search for more ap-
between 2.65 and 2.75 inches (to allow for filter propriate solutions will continue, if for no other
load up) over the range of 0 to 150 ft3/min is reason, because of the ever-shifting balance be-
optimal. Since the demand of one cage rack is tween the conflicting demands that (i) personnel
only 15 ft3/min, one such blower can serve up to be protected from even possibly infectious organ-
10 cage racks without need for controls. isms; (ii) the scientist's access to his experimental
(viii) Use with biological safety cabinets. In some animals (and hence the speed at which he can
cases, the etiological agents being used are of such work) be hampered no more than absolutely nec-
pathogenicity or toxicity that the animals must be essary; (iii) extraneous biological or toxic con-
inoculated or exposed within a negative-pressure tamination be excluded; and (iv) the maximal
gas-tight cabinet. To transfer the animals to the number of animals be held in the space available
ventilated isolation cage in such instances, the accompanied by the minimal possible capital
cabinet is fitted with a transfer cylinder (in its outlay for equipment and maintenance.
floor) identical in size to the cage feed port neck. The approach to the design of the ventilatedVOL. 16, 1968 NEW ISOLATION CAGE 771
isolation cage reported herein was directed toward isolation of infected animals in a single room.
developing a useful tool for scientific research, J. Bacteriol. 40:569-580.
emphasizing aerodynamic precision, minimal 8. Kethley, T. W., and W. B. Cown. 1966. Disposi-
maintenance, and adaptability for different levels tion of airborne bacteria in clean rooms. Ann.
of hazard and contamination control. Technical Meeting, American Assoc. Con-
On a first cost basis, the economics of holding tamination Control, 5th, Houston, Tex.
9. Phillips, G. B., and J. V. Jemski. 1963. Biological
animals in the ventilated isolation cage is compa- safety in the animal laboratory. Lab. Animal
rable to holding them in plastic isolators, and Care 13:13-20.
many more can be maintained per square foot of 10. Phillips, G. B., F. E. Novak, and R. L. Alg. 1955.
floor area. Portable inexpensive plastic safety hood for
Reversal of air flow through the cage, i.e., push- bacteriologists. Appl. Microbiol. 3:216-217.
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ing instead of pulling, results in air flows and 11. Phillips, G. B., M. Reitman, C. L. Mullican, and
pressures equal in magnitude but opposite in di- G. D. Gardner. 1957. Applications of germicidal
rection, thus suggesting possibilities of use for ultraviolet in infectious disease laboratories.
III. The use of ultraviolet barriers on animal
breeding and rearing highly defined, if not germ- cage racks. Proc. Animal Care Panel 7:235-244.
free, animals. Other possible uses are for hypo- 12. Phillips, G. B., and R. S. Runkle. 1967. Laboratory
baric studies and in the area of maintaining special design for microbiological safety. Appl. Mi-
environments such as 02, N2, or others. crobiol. 15:378-389.
13. Rauscher, F. J., L. M. Carrese, and C. G. Baker.
ACKNOWLEDGMENTS 1966. Survey of viral oncology with particular
I acknowledge the guidance and encouragement of reference of lymphomas. Cancer Res. 26:1176-
R. S. Runkle, A. S. Gates, C. D. LeMunyan, C. T. 1184.
Hansen, S. E. Stewart, N. H. Wiebenga, W. T. Hann, 14. Reyniers, J. A. 1959. Design and operation of
and L. G. Herman, and the help of Elmer Dyson, apparatus for rearing germ-free animals. Ann.
who ably assisted in aerodynamic testing, and Carl N.Y. Acad. Sci. 78:47-79.
Schumacher, who conceived the clamping mechanism. 15. Reyniers, J. A., and P. C. Trexler. 1943. The
germ-free technique and its application to
rearing animals free from contamination, p.
LITERATURE CITED 114-143. In J. A. Reyniers [ed.], Micrurgical
1. Ahlstrom, C. G., and N. Forsby. 1962. Sarcomas and germ-free methods, Charles C Thomas,
in hamsters after injection with Rous chicken Publisher, Springfield, Ill.
material. J. Exptl. Med. 115:839-852. 16. Smadel, J. E. 1951. The hazard of acquiring virus
2. Bedson, S. P. 1940. Virus diseases acquired from and rickettsial diseases in the laboratory. Am.
animals. Lancet 2:577-578. J. Public Health 41:788-795.
3. Decker, H. M., B. S. Buchanan, M. S. Hall, and 17. Stewart, S. E., and B. E. Eddy. 1958. A review of
B. S. Goddard. 1962. Air filtration of microbial the biological properties of S E polyoma virus.
particles. Public Health Serv. Publ. #953. Proc. Intern. Congr. Intern. Soc. Hematol., 7th,
4. Gremillion, G. G. 1959. The use of bacteria-tight Rome.
cabinets in the infectious disease laboratory. 18. Stewart, S. E. and J. C. Landon. 1964. Hamster
Proc. Symp. Gnotobiotic Technology, 2nd, p. tumors induced with Rous virus (Bryan strain)
171-182. Univ. of Notre Dame Press, Notre "activated" with a factor from rapidly growing
Dame, Ind. tissues. Natl. Cancer Inst. Monograph 17, p.
5. Harstad, J. B., and H. M. Decker, L. M. Buch- 237-255.
anan, and M. E. Filler. 1967. Penetration of 19. Trexler, P. C., and L. I. Reynolds. 1957. Flexible
submicron TI bacteriophage aerosols and bac-
terial aerosols through commercial air filters. film apparatus for the rearing and use of germ-
Proc. Ann. Technical Meeting and Exhibit, 6th, free animals. Appl. Microbiol. 5:406-412.
p. 200-204. American Association for Con- 20. U.S. Government Printing Office. 1956. Filter
tamination Control, Washington, D.C. units, protective clothing, gas-mask components
6. Harstad, J. B., H. M. Decker, M. E. Filler, and and related products: performance-test meth-
C. R. Phillips. 1967. Evaluation of air filters ods. Military Standard 282, Washington, D.C.
with submicron viral aerosols and bacterial 21. U.S. Public Health Service. 1963. Guide for
aerosols. Department of the Army, Ft. Detrick, laboratory animal facilities and care. Public
Frederick, Md. Final Report, Interagency Health Serv. Publ. S 1024 (1965 ed.).
Service Agreement MIPR 6.0037 with National
Cancer Institute, National Institutes of Health, 22. Wedum, A. G. 1954. Laboratory safety in research
p. 1-37. with infectious aerosols. Public Health Rept.
7. Horsfall, F. L., and J. H. Bauer. 1940. Individual U.S. 79:619-633.You can also read
