Moreover_ to operate a CO2 cabinet in a closed cold room is

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					AN IMPROVED C02 CABINET FOR LOW TEMPERATURE STORAGE
   OF INFECTIOUS AGENTS WITH GASEOUS CO2 EXCLUDED
           FROM THE SPECIMEN COMPARTMENT
             FRANK L. HORSFALL, JR., AND HAROLD S. GINSBERG
     The Hospital of The Rockefeller Institute for Medical Research, New York, N. Y.
                       Received for publication January 11, 1951
   The development of a low temperature storage cabinet for the preservation
of viruses and other infectious agents some years ago (Horsfall, 1940) led to the
use of similar solid C02 cabinets in numerous laboratories. The original cabinet
has been in continuous operation for more than 11 years, and others more or less
like it have been operating for almost as long. Despite the generally satisfactory
results obtained by many workers with solid CO2 refrigerators of this kind, the
use of the cabinet presents certain problems. Prominent among these are the
following: (1) a decrease in the pH of unsealed specimens as a result of the
absorption of gaseous C02, and (2) the cost of operation due to rapid consump-
tion of solid CO2. Although a decrease in pH can be prevented by sealing speci-
mens in glass or by the addition of an adequate buffer, neither practice is entirely
satisfactory. Specimens sealed in glass are inconvenient to handle, and glass
containers may break as a result of the marked temperature change (-i100 C)
that occurs during rapid freezing or thawing. Buffers tend to fix the ionic en-
vironment of an agent in a manner that may preclude desired manipulation of
this variable. Moreover, the addition of a buffer results in dilution of the in-
fectious agent. The rate of consumption of solid CO2 can be reduced by increasing
the efficiency of the insulation used and by decreasing the external temperature
of the cabinet. However, to increase the thickness of the insulating material
much beyond 6 inches causes the cabinet to become unwieldly and inconvenient.
Moreover, to operate a CO2 cabinet in a closed cold room is dangerous for
laboratory personnel unless adequate provision is made for exhausting from the
room the large amounts of CO2 gas which evolve.
  With a view to overcoming the problems mentioned above, a solid C02 cabinet
of markedly different design was developed in this laboratory 2 years ago. The
cabinet has been in continuous operation during this period and has proved
entirely satisfactory. Certain novel features in design contribute to the advan-
tages of the improved cabinet: (1) gaseous CO2 is entirely excluded from the
specimen cabinet, and, as a consequence, no change in the pH of unsealed and
unbuffered specimens occurs; (2) the cabinet operates in a closed cold room held
at a temperature of 4 C and, as a result, the consumption of solid C02 is less
than half what it would otherwise be; (3) all gaseous C02 from the refrigerant
bunkers is exhausted outside the cold room, and there is therefore no personnel
hazard from operating the cabinet in a closed space.
  Desin of the cabinet. The details of the design of the cabinet' are shown in
   1 The cabinet was constructed by Gardner-Neilsen, Inc., 21-38 44th Road, Long Island
City, New York, from specifications supplied by this laboratory.
                                          443
444           FRANK L. HORSFALL, JR., AND HAROLD S. GINSBERG               [VOL. 61
figures 1 and 2. The cabinet is of double wall construction and is insulated with
cork board applied and sealed in hot asphalt. The front wall, top, and door
covers are made of stainless steel, 20 gauge. The inner walLs, bottom, compart-
ment partitions, cooling vanes, and door bottoms are made of stainless steel,
18 gauge, and all joints are soldered gas-tight. In effect, the inner walLs, bottom,
top, and doors form a gas-tight tank that is separated into three gas-tight com-
partments by two vertical sheets of stainless steel (figure 2,b). Two compart-




                Figure 1. Front and top views of improved C02 cabinet

ments, one at either side (figure 2,c) are solid C02 bunkers, and the large central
compartment (figure 2,d) between the bunkers is the specimen compartment,
which contains 40 vertical stainless steel drawers, each of which is equipped
with specimen shelves. Under the entire bottom of the inner tank there is an
k-inch steel plate for reinforcement.
   Because the cabinet was designed to be built in against the inner walls of a
cold room, the walls of which are insulated with 6 inches of cork board in asphalt,
the thickness of the insulation between the outer and inner wals of the cabinet
itself varies as can be seen in figure 2. The front wall contains 6 inches of insula-
1951]             LOW TEMPERATURE STORAGE OF INFECTIOUS                AGENTS         445
tion, the back wall 5, the two side walls and the bottom 2, and the doors 3. The
only surfaces of the cabinet that are in contact with the air of the cold room
(4 C) are the front, top, and doors. The sides, bottom, and back form an integral
part of the cold room itself and therefore have a total thickness of cork board
insulation of 8, 8, and 11 inches, respectively, separating the inner steel tank
from the surrounding rooms.
   One large door (figure 1,A), hinged at the back and counterbalanced with 70
pounds of lead, allows access to the specimen compartment. Each C02 bunker is
                  5ection thpough top
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                  5ection      thpough                 front




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                            A                                           Section A-A
   Figure 2. Diagrammatic sections through top, front, and side of cabinet to show details
of design. Note protruding plastic pipes for discharge of C02 gas from C02 bunkers.

covered by a smaller but similar door (figure 1,B). The central area of the top
surrounding the door to the specimen compartment is 1 inch higher than the
lateral areas of the top around the bunker doors. This construction detail was
incorporated so that cold C02 gas, which is denser than air, would not flow by
gravity from an open bunker into the open specimen compartment should the
doors to both inadvertently be opened together. A fixed rule interdicts the
opening of the compartment if either bunker door is open.
  The dimensions of the cabinet in inches as illustrated in figure 2 are: (1) out-
side-length 71, width 363, height 40; (2) inside over-all-length 67, width
254, depth 35; (3) specimen compartment-length 43, width 251, depth 35;
(4) C02 bunkers, each-length 12, width 25j, depth 34; (5) specimen compart-
ment door-length 39k, width 271, thickness 3; (6) C02 bunker doors, each-
446           FRANK   L. HORSFALL, JR., AND HAROLD S. GINSBERG            [VOL. 61
length 131, width 271, thickness 3; and (7) cooling vanes-one central vane
(figure 2,e), width 251, height 28; and eight lateral vanes (figure 2,f), length
21k, height 28. The lateral cooling vanes are parallel, spaced 5 inches apart, and
are soldered to the bottom, the side bunker walls, and the central vane. The
central vane is soldered to the front and back walls of the specimen compartment
as well as to the bottom. The vanes are solid sheets of stainless steel, gauge 18,
and are not perforated.
   The top is designed to fit very tightly around all outer surfaces of the upper
part of the inner steel tank and is pressed on firmly in hot asphalt. The top is
constructed on a heavy oak frame, the sides of which overlap the steel walls of
the inner tank by 1 inches. Above the inner steel walls of all three compartments
and overlapping them by 1 inch is a strip of bakelite, 6 inches wide, which is
tightly screwed to the oak frame and serves to hold the upper 1 inches of the
steel walls in close contact with the frame of the top. Between the framework
and the stainless steel covering of the top is 3 inches of cork board insulation in
asphalt.
   The doors overlap the top by 1 inch in all dimensions. All the doors are pro-
vided with {-inch sponge rubber gaskets, i inch in width, which on closing meet
similar gaskets cemented to the top of the cabinet and provide gas-tight seals.
The hinges, handles, and locks for the doors are chrome-plated brass. The
cabinet operates inside a cold room, 6 feet by 7 feet 41 inches by 9 feet 10j inches
in height, which is kept at 4 C mechanically. The humidity of the air of the cold
room is reduced with large trays of CaC12, which is replenished each week. All
C02 gas is exhausted from each bunker through a 11-inch plastic pipe that
extends entirely through the side wall of the cabinet and the cold room, is
covered by a hinged plastic flap valve, and vents into the rooms adjacent to the
cold room. Each C02 bunker can hold six unbroken 50-pound cakes of solid C02;
the total capacity of the two bunkers is 600 pounds of CO2. Canvas-covered
blankets (figure 2,g), provided with canvas handles at the corners and filled
with a 3-inch layer of kapok, are used in the specimen compartment and each of
the C02 bunkers. These fit snugly between the walls of each compartment and
provide insulation in addition to that afforded by the doors.
   The details of the design of one of the 40 specimen drawers are shown in
figure 3. The drawers are made entirely of stainless steel, gauge 24. Their outside
dimensions in inches are: height 28, width 41, and depth 4j. The total storage
capacity of all the drawers is 14.05 cubic feet. Three shelves, at an angle of 30°
from the honrzontal, are riveted to the sides 2, 13, and 20 inches from the lower
end of the drawer (figure 3,A). Removable shelves of two sizes, which also lie in
the drawer at an angle of 300, are provided. The larger shelves (figure 3,B)
provide surfaces 11 inches apart, the smaller (figure 3,C), j inch apart. There is
space for 16 large shelves or 28 small shelves in each drawer, and any desired
combination of the two types may be made. It has been found that an efficient
distribution is 7 large shelves in the lower part of the drawer and 16 small shelves
above them. Because of the angle of the shelves, specimen containers do not fall
out of the drawer when it is raised or lowered and, because of the position of the
1951]        LOW TEMPERATURE STORAGE OF INFECTIOUS AGENTS                             447
cooling vanes, each drawer is completely closed when resting on the bottom of
the specimen compartment.
  Nitrocellulose tubes with metal screw caps similar to those described pre-
viously (Horsfall, 1940) are employed.2 These are in three sizes: small, 31 inches
by i inch with a capacity of 8 ml; medium, 4 inches by 3 inch with a capacity
of 20 ml; and large, 41 inches by 7 inch with a capacity of 40 ml. The total
number of specimens that can be stored in the 40 drawers depends obviously
upon the types of shelves and tubes used. With small shelves 7 inch apart and
7 small tubes per shelf, the capacity of the drawers is 7,840 tubes that contain




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                AA




                                   -B
                At


    Figure S. Detail of one specimen drawer with large and small removable shelves.

62.8 liters. With small shelves and 5 medium tubes per shelf, the capacity of the
drawers is 5,600 tubes that contain 112 liters. With large shelves 11 inches
apart and 4 large tubes per shelf, the drawers can hold 2,560 tubes that contain
102.4 liters. As used in this laboratory with 7 large and 16 small shelves per
drawer, with small tubes only on the small shelves, and with medium and large
tubes in equal numbers on the large shelves, the capacity is 5,740 tubes that
hold 73.6 liters. With all but the three fixed shelves removed, large containers
may be stored in a drawer; two of the spaces thus produced have dimensions
of 71 inches by 4j inches by 41 inches, and the third has dimensions of 11 inches
by 42 inches by 4' inches.
  2 Tubes and caps are manufactured by the Lusteroid Container Co., Inc., 10 Parker
Avenue West, Maplewood, New Jersey.
448              FRANK L. HORSPALL, JR., AND HAROLD S. GINSBERG                     [VOL. 6 1
  A detailed drawing of a drawer as usually employed in this laboratory with
some tubes of each size in place is shown in figure 3. Details of the shelf design
are also shown in two side views (figure 3,D) at different angles. For conveni-
ence in filing and finding specimens, each drawer handle has a number stamped
into the metal.
   Solid C02 consumption. The two bunkers have a total capacity of 600 pounds
of solid CO2. On the average, 50 pounds of solid C02 evaporates from the cabi-
net per day. Thus, in approximately 6 days, half of the C02 in the bunkers is
consumed. The temperature of the specimen compartment does not increase
significantly if the bunkers are but half-filled, and therefore C02 need not be
added frequently. At times, periods of 5 days have been allowed to elapse be-
tween additions of C02, and no definite increase in temperature has occurred.
                                         TABLE 1
               Temperature and concentration of C02 in specimen compartment
                                               SPECIMEN COMdPARTMENT
  MONTHS OF CONTINUOUS
  OPERATION OF CABINET
                              Temperature              COs               pH of watert
                                   C                 volume %
           1                      -65                                         6.0
           2                      -62                                         6.4
           6                      -62                  0.23                   6.3
           9                      -65                  0.10
          13                      -60                  0.13
          16                      -62                  0.13
          22                      -62                  0.20
   * Thermometer in center of specimen compartment as distant as possible from           C02
bunker walls.
   t Stored in uncapped nitrocellulose tubes.
In general, a regular schedule is followed, and additional refrigerant is added
on Monday, Wednesday, and Friday. The efficiency is such that only 3.53
pounds of C02 per day are consumed per cubic foot of storage drawer space.
This figure may be compared with 7.96 pounds per day per cubic foot of drawer
space for the original cabinet (Horsfall, 1940) and represents a decrease of 56
per cent in the consumption of C02 per unit volume with the present cabinet.
   CO2 concentration and temperature. A summary of data regarding the concen-
tration of CO2' in the air of the specimen compartment as well as the tempera-
ture in the compartment is given in table 1. Neither the C02 concentration, which
has been in the vicinity of 0.20 per cent or less, nor the temperature, which has
ranged between -60 and -65 C, has varied appreciably during 22 months of
continuous operation. On the possibility that C02 gas might at times accidentally
gain access to the specimen compartment and remain there indefinitely because
of its density, one open tube containing solid soda lime is kept on the lower shelf
of each drawer. This is done despite the fact that there is some doubt that soda
  3Dr. James R. Weisiger kindly carried out the gasometric analyses for C02.
1951]              LOW TEMPERATURE STORAGE OF INFECTIOUS AGENTS                            449
lime will react with C02 at this low temperature. As is also shown in table 1,
open tubes of distilled water, frozen and stored in the cabinet for as long as 6
months, have shown no decrease in pH. Similar tubes of distilled water, which
were stored in a cabinet of the original type without C02 exclusion, had a pH of
5.3 after 1 week and 4.6 after 2 weeks of storage. Normal allantoic fluid, stored
under similar conditions without exclusion of C02, had an initial pH of 7.7;
after 1 week the pH was 6.2.
  Sterilization of tubes. Although ultraviolet irradiation may be used to sterilize
nitrocellulose tubes as indicated previously (Horsfall, 1940), it has proved to be
much more convenient to sterilize them by prolonged heating at 68 C. For 2
years the following procedure has been employed and found to be entirely
                                     TABLE 2
  Maintenance of constant infectivity titers of various viruses during prolonged storage
                                                    VIRUSE8*

 MONTHS O1 STOI-       PR8          Lee         |   Mumps                 PVM
 AGE IN CABnINT
                                                                        Log M.S4,
                                Log. E.I.D.,,
                                                                  (a)                (b)
         0             -7.3         -9.0             -7.2        -4.9               -3.8
         2             -7.7         -8.7             -6.7        -4.8
         4             -7.3         -8.7             -7.0
         6                                                       -4.9
         8             -8.0        -9.0
        12             -7.5        -8.5              -6.9                           -4.6
        16             -7.5        -8.7              -7.2
   * All viruses, except PVM, were contained in infected allantoic fluid to which nothing
was added. PVM (a) was a 10 per cent mouse lung suspension in 10 per cent normal horse
serum broth. PVM (b) was a 10 per cent mouse lung suspension in distilled water. Each
preparation was stored in nitrocellulose tubes that were not sealed.
satisfactory: Nitrocellulose tubes are filled with dilute "duponal" solution and
allowed to stand in it for 3 hours. They are then rinsed 6 times under runnng
tap water, filled with tap water, held for 24 hours, and then rinsed 3 times with
distilled water. They are then inverted in wire racks and allowed to drain until
dry. After this, the racks and tubes are placed in a steam-heated cabinet that
is kept constantly at 68 C, and they are held at this temperature for 48 to 72
hours. A metal screw cap that has been sterilized in the autoclave is then placed
on each tube. Repeated tests under aerobic and anaerobic conditions have con-
stantly failed to reveal the presence of viable bacteria in tubes sterilized in this
manner.
  Maintenance of viral infectivity. A summary of the results of successive in-
fectivity titrations with various viruses stored for considerable periods in the
cabinet is shown in table 2. PR8, Lee, and mumps viruses were stored purposely
in unsealed nitrocellulose tubes as undiluted infected allantoic fluid. Because
of the low buffer capacity of allantoic fluid, marked reduction in pH occurs
450           FRANK L. HORSFALL, JR., AND HAROLD S. GINSBERG              [voL. 61
within a few weeks in the original type of C02 cabinet, and the infectivity
titers of viruses in allantoic fluid may decrease rapidly unless material with a
high buffer capacity is added or unless the specimens are sealed in glass. It is
evident that no significant change in infectivity titer occurred in the three
viruses employed during 16 months of storage in the present cabinet. With
PVM, similar results were obtained during 12 months of storage. A considerable
number of other viruses, including herpes simplex, Newcastle disease, the
GDVII and FA strains of mouse encephalomyelitis, various types of Coxsackie,
and different strains of poliomyelitis, as well as a number of strains of the en-
cephalitis group, have been stored under similar conditions for varying periods.
In no case has there been any demonstrable decrease in infectivity titer.
                                   DISCUSSION
   The improved solid C02 cabinet described in this communication was designed
to overcome certain disadvantages inherent in the earlier type of cabinet (Hors-
fall, 1940). Because gaseous C02 is effectively excluded from the specimen com-
partment as a result of gas-tight construction, there is no need to seal specimens
or add buffers; reduction in pH of unsealed and unbuffered specimens does not
occur. Transfer of heat from frozen specimens to the refrigerant bunkers is
accomplished by conduction through sheet stainless steel and to some extent by
radiation. Cold air in the specimen compartment probably plays little or no role
in the thermal properties of the cabinet, and there is no gas exchange between
the refrigerant bunkers and the specimen compartment. Because of the effi-
ciency of the insulation employed and the reduction in the outside temperature
of the cabinet which results from installing it in a cold room at 4 C, it uses only
44 per cent as much solid C02 per unit volume of storage space as did the origi-
nal cabinet (Horsfall, 1940).
   During 2 years of continuous operation the present cabinet has proved to be
entirely satisfactory and maintains specimens at a temperature of -60 to
 -65 C. How long constant infectivity titers of unstable viruses may be main-
tained in the cabinet is not yet known. Inasmuch as no change in the concentra-
tion of infective virus occurred with four relatively unstable agents during 12
to 16 months, it seems probable that most infectious agents could be stored for
long periods without the risk of inactivation. Contrary to anticipation, the
accumulation of ice in the specimen compartment has been very slow. Only
after 20 months of operation was it necessary to remove a layer of finely granu-
lar ice from the bottom of each of the drawer spaces. It should be emphasized
that if ice is allowed to accumulate sufficiently to impede heat transfer from the
specimen drawers to the bottom, side walls, and cooling vanes, a significant rise
in the temperature of the specimen compartment may occur. In addition, when
ice accumulates in the kapok blankets covering the bunkers and the specimen
compartment, the blankets should be replaced with others free of ice. The origi-
nal blankets may then be thawed and thoroughly dried in a hot-air oven.
1951]        LOW TEMPERATURE STORAGE OF INFECTIOUS AGENTS                      451

                                    SUMMARY
   Carbon dioxide gas is excluded from the specimen compartment of a solid
C02 storage cabinet of improved design which is described herein. Because of
this feature, the pH of specimens does not decrease, and neither sealing in glass
nor the addition of buffer is necessary. Unstable viruses stored in the cabinet
under these conditions maintained constant infectivity for long periods. A
marked decrease (56 per cent) in consumption of solid C02 is effected by efficient
insulation and the installation of the cabinet in a refrigerated room.
                                   REFERENCES
HORSFALL, F. L., JR. 1940 A low temperature storage cabinet for the preservation of
   viruses. J. Bact., 40, 559-568.

				
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