following distribution into ampoules led to the development of the freezing

MACHINE FOR SHELL FREEZING SM\IALL VOLUMES OF BIOLOGICAL PREPARATIONS L. D. ANTOINE AND M1. V. IIARGETT United States Public Health Service, Rocky illountain Laboratory, IIamnilton, Montana Received for publication, Jtuly 26, 19.43 The preferred method for preserving certain types of biological preparations, such as yellow fever vaccine, consists in quick-freezing followed by desiccation under high vacuum from the frozen state with subsequent storage in vacuo or an atmosphere of dry nitrogen (Sawyer ct al., 1929; Bauer ct al., 1940; Hargett et al., 1943). In the Rocky MNIountain Laboratory of the United States Public Health Service, yellow fever vaccine is put up in pyrex ampoules of the type shown in figure 1. The stem of the ampoule measures 7 by 60 mm. and the body 25 by 90 mm. Although ampoule capacity approximates 28 ml., vaccine content is limited to 5 ml. This volume relationship was established to permit the rapid freezing of the vaccine in the form of a thin-walled hollow cylinder preparatory to desiccation. This method of freezing has been denoted "shell freezing." Ampoules of similar design and reduced capacity are employed to receive lesser volumes. The requirement of a rapid and efficient means of shell freezing the vaccine following distribution into ampoules led to the development of the freezing machine to be described. Efficiency, availability, and cost factors dictated the employment of carbon dioxide ice as the refrigerant and 95 per cent ethyl alcohol as the circulation mediuim. THE MACHINE Because of the difficulty of obtaining metal and parts, brought about by war requirements, construction was of locally available materials. An alcoholresistant paint replaced the to-be-desired chromium plating. Construction was of metal with cork insulation. One photograph (fig. 1) and two drawings (figs. 2 and 3) show general construction and most details. The unit consists of dry ice chamber A, freezing compartment B, ampoule rotating unit C, ampoule leveling device D, and pump E. The dry ice compartment is closed by snug-fitting cover A,. Vent A2 leads from near the top of ice chamber for exhausting carbon dioxide while alcohol is emptied by drain line A3. The escape of dry ice particles by way of fluid openings in compartment A is prevented by fixed screens. The carbon dioxide ice is contained in wire basket A4. The depth of the freezing compartment B is 3' inches compared to 9' inches for dry ice chamber A. Refrigerated alcohol is received into B from line E4 and directed to remote portions of chamber by baffles B1 and B2. Alcohol is returned to compartment A from chamber B through the duct formed by the walls of the partition common to A and B. Slot-shaped duct inlet 133 is shown in figures 1 525

526 L. D. ANTOINE AND M. V. HARGETr and 3. The lower lip of the duct inlet is adjustable to permit some alteration of channel mouth. The duct outlet, not shown in figures, is a slot-shaped opening at bottom of chamber A. Retrograde passage through the duct of carbon FIG. 1. "s '1I AMPOULE FREEZING MACHINE WITH ONE 28 ML. AMPOULE IN FREEZING POSITION CALIK -U AMPULE FREMG PJOCHINr hsv.uad g.ddw. w L.D. J0fe _ 4-o FIG. 2. ToP VIEW TO SHOW GENERAL CONSTRUCTION Note that long axes of ampoules are slightly non-parallel to drive wheel shafts.

SHELL FREEZING BIOLOGICAL PREPARATONS dioxide evolved in A is prevented by the trap construction of the duct. The small quantity of fluid lost through leveling device notches D2 is collected in trough B4 and caried away through drain B5. In emptying the machine of alcohol, compartment B is drained by line Bo. Ampoule rotating unit C consists of 9 rubber-tired drive wheels Ci directly connected by shafts to gear train C2 which is driven by constant speed fractional horsepower motor Co via shaft and drive gear C4. Idler gears are employed so that all drive wheels rotate in the counterclockwise direction. The motor is A_vSCALE INWCHES Sec..X*R FIG. 3. VERTICAL CROSS SECTION THROUGH FREEZING CHAMBER AND Dmvam MOTOR fixed to the backside of compartment B, while the drive mechanism is mounted on a chassis secured by brackets within freezing chamber B. Screws at both ends of the chassis permit alignment and leveling. Plate C5 serves to retain the butt ends of the ampoules which are forced against it by. the fact that the axes of the rotating ampoules are slightly non-parallel with the axes of the drive wheel shafts. This is obtained by notches D2 being placed slightly to the right of a point in line with the axis of the opposite idler gear shafts. The plate is mounted on pivots allowing it to be turned front or back to accommodate 527

528 L. D. ANTOINE AND M. V. HAGETT ampoules of varying body length. Figure 1 shows it in the "back" position. Protective guards, which are readily removed, hield drive motor and gears. Ampoule leveling device D is mounted on front of compartment B. It supports ampoule stems and serves to level ampoules of varying body diameter by elevating or lowering the stems. Fixed plate DI contains eight notches D2 whose relation to the drive mechanism has been described. Notch margins have beveled edges to reduce drag on rotating ampoules. Movable plate D3, whose upper edge is beveled, fits snugly on the front side of fixed plate DI and is secured to it by flat-headed pins at either end, passing through slots which permit vertical movement. Adjustment of Ds is effected by shaft Di fitted with fixed pinion gears meshing with gear teeth in the movable plate. The shaft is activated by small worm drive Dr, operated by a thumb screw. Centrifugal pump E, mounted on backside of compartment A, is powered by a variable speed motor E1 controlled by a rheostat switch E2. Pump intake line E3 takes off from near the bottom of chamber A, while pump discharge line E4 terminates within compartment B. This latter line is insulated by a thickwalled snug-fitting rubber jacket. The machine is fixed in position on a laboratory bench by a threaded steel rod secured to the middle of the underside of the unit and passing into the bench. Adjustment screws on each of the four legs permit ready leveling. A rubber tube attached to vent As conducts carbon dioxide to the floor. Additional tubes connecting to alcohol drain lines AL and B]3 pass to a collection bucket under the bench. An electric fan is mounted on the wall in a horizontal position directly above the machine to direct a current of air onto the unit. To place machne in operation, basket A4 within compartment A is almost filled with chunks of carbon dioxide ice. Pump E is then set in motion and pre-chilled (minus 600C. to minus 750C.) alcohol added so that the fluid level in A is about two inches below the inferior margin of notches D2. Cover A1 is then placed in position to prevent overflow of generated carbon dioxide gas from A. The fan is then turned on to force the carbon dioxide evolved from the alcohol in chamber B to the floor, from whence it is removed by the room exhaust. Removal of carbon dioxide from the vicinity of the freezing chamber is desired to prevent its being drawn into ampoules during freezing. Alcohol, refrigerated by intimate contact with dry ice in A, is drawn through line Es to the pump and impelled via line E4 to freezing compartment B. Return to dry ice chamber is by gravity via channel B3. Temperature in the freezing chamber is to an extent regulated by flow from A, which is governed by the operational speed of the pump. A temperature of minus 600C. to minus 700C. is readily maintained in the freezing compartment. Height of alcohol in B is so fixed that the inferior 4 to 9 mm. of ampoules in freezing position are immersed. This is accomplished by adjustment of the lower lip of channel mouth Bs and ampoule rotating unit C. As many as eight ampoules of the type described, loaded with vaccine and plugged with cotton, are placed in the machine as illustrated with bodies supported by drive wheels and stems by properly elevated leveling device D. The

SHELL FREEZING BIOLOGICAL PREPARATIONS5 drive mechanism rotates the ampoules in a clock-wise direction at 116 R.P.M. This speed of rotation insures a constant film of cold alcohol on the non-immersed portions of the ampoules. The 5 ml. of contained vaccine solidly freezes in the form of a thin-walled hollow cylinder, suitable for desiccation, in about 15 seconds. As freezing is completed the ampoules are removed to a holding bath of dry ice and alcohol. Similar style ampoules of varying sizes and content may be "shelled" with equal ease. At termination of freezing, cover A1 is removed and dry ice taken out by means of container basket A4. Alcohol is drained via line A3. COMMENT Employing the 28 ml. ampoules (fig. 1) containing 5 ml. of vaccine each, one operator with the described machine can freeze 1,000 units per hour. The resultant frozen shell is far more perfect and suitable for desiccation than was vaccine frozen by previously employed hand methods. The use of a friction rather than an engagement drive for rotation of ampoules simplifies and accelerates operation. SUMADRY One operation in the desiccation method of preserving certain biological preparations, such as yellow fever vaccine, requires that it be frozen rapidly in the form of a thin-walled hollow cylinder. The electrically operated machine described, employing carbon dioxide ice as the refrigerant and ethyl alcohol as the circulation medium, accomplishes this effectively and rapidly, with only a single operator required. REFERENCES BAUER, JOHANNES H., AND PICKELS, EDWARD G. 1940 Apparatus for freezing and drying virus in large quantities under uniform conditions. J. Exptl. Med., 71, 83-88. HARGETr, M. V., Buiwuss, H. W., AND DONOVAN, ANTHONY 1943 Aqueous-base yellow fever vaccine. U. S. Pub., Health Repts., 58, 505-512. SAWYER, W. A., LLOYD, W. D. M., AND KITCEN, S. F. 1929 The preservation of yellow fever virus. J. Exptl. Med., 50, 1-13. 529