Equipment for Cultivation of Microorganisms c.-G. H E D ~ NAND B. MALMGREN Bacferiologicaf Departmenf, Karofinska lnsfifutef Medical School, Stockholm, Sweden
E
QUIPMEKT specially designed for sterile handling of large quantities of biological material is of growing importance to a number of industries. The need for this equipment exists not only in fermentation plants but also in those branches of the pharmaceutical industry where the products are intended for injection into the human body-serums, vaccines, plasma substitutes, etc. I n addition, a number of bacteriological research laboratories have programs which call for large scale production of microorganisms, which means that they have important sterility problems, particularly when they are studying pathogenic bacteria. I n the case of production units which are erected to operate unchanged throughout the years, the usual method of transferring the sterile product from one apparatus to another is by a rigid system of -DiDes - and valves. I n order to be satisfactory from a bacteriological point of view, however, an arrangement of this kind generally calls for steam locks a t all critical points, and the setup often becomes a maze of valves and pipes for live and condensed steam. The system often becomes costly and difficult to operate; more important, however, it is difficult to keep clean, particularly the elbows, joints, and corners. MoreOver materials, that are deleterious to the biological product sometimes have t o be used for gaskets, membranes, etc.
A
8 C
I
0 E
J
F G
H
Plug for Closing
Coupling and Connector Plug
Figure 1 . A. 8.
C, D.
E. F. G.
Flexible Tube Connection H. Rubber valve
Flexible tube Screw cap with hose opening Central portion of spring-loaded hollow plug Spring-loaded ring Steam groove Steam inlet Adaptable pipe extension for opening rubber valve
September 1954
I.
While these drawbacks may not be so important in the factory, they are indeed a major disadvantage in a pilot plant for a high degree of flexibility is also necessary. The process may have t o be changed from one day to another, and it is desirable that it should be possible t o make such change with a minimum of qualified assistance from the workshop. The systcm described in this paper has been developed in order to allow a maximum of flexibility in a pilot plant without jeopardizing the sterility of the material which is handled. The system has been designed to operate with any desired microorganism, nonpathogenic as well as pathogenic. Use of Flexible Tubing Offers Advantages in Operation,cleanup,and Choice of Materials
The basic assumption &as that most of the disadvantages of a rigid pipe system can be avoided if flexible tubes are used. The use of flexible tubing is feasible in the biological pilot plant, because, as a rule, operating pressures are low. Hoses offer the advantage that the valves can be replaced by hose clips, and the liquid can easily be pumped by means of ordinary hose pressure pumps. d considerable simplification can thus be achieved, not only in operation but also in cleaning. The price of ordinary flexible tubing is actually so low that an exchange of tubes may often be preferred to a thorough rinsing. I n view of the fact that the sensitivity of different biological products to toxic ions, pH, etc., varies, it is necessary t o choose with care the K materials with which they will come into contact. The great number of rubber and plastic materials L available in the form of tubing offer a wide range from which to choose. However, sometimes a M material must be used that does riot stand up to the internal steam pressure required to achieve sterilization, either because it is too soft or because it undergoes a chemical change. In some cases, it might be possible to reinforce the tubing by a surrounding wire net or a metal pipe, but often the tubing is not sterilized in situ but in an autoclave, or possibly in an atmosphere of ethylene oxide (5). Consequently, facilities must be arranged for attaching sterile tubing to an apparatus that has been sterilized separately. Socket The points of attachment should afford possibilities for the alternative use of metal piping or flexible tubing.
K.
Funnellike element with inner conical surface Steam outlet Screw cap without hose opening
1.
Spring-loaded ring
J.
M. Central portion of spring-loaded solid plug
Specially Designed Connections Permit Easy Assembly and Sterilization
The most important problem that had to be solved in order to permit the use of tubing was t o find a suitable device for the sterile connection.
INDUSTRIAL AND ENGINEERING CHEMISTRY
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ENGINEERING, DESIGN, AND PROCESS DEVELOPMENT
Figure 2.
100-Liter Fermentor with Two Flexible Tube Connections Attached to Lid
The simplest method-pushing a flexible tubing over an ordinary pipe socket-cannot be used, since the tubing might slip off, especially if the internal pressure was increased, and also because microorgariisnis could groiv through the capillary space betneen the tubing and the piping. The latter risk could be eliminated by making the socket either wholly or partly steam-jacketed. I n this case, however, the socket often becomes so tightly wedged that it can only be removed by cutting. If the tubing is pulled off, it is practically impossible to avoid splashes and this is very dangerous when a culture of pathogenic bacteria is being handled. Besides, i t is exceedingly difficult to put sterile tubing on a pipe socket in a way that is satisfactory from a bacteriological point of view, particularly if the socket is hot. Funnellike elements shown in Figure 1 ( 1 )are welded to pieces of equipment which are to be connected (Figures 2, 3, and 4 ) , as well as to each other, in order to allow for extension and branching of the tubing (Figure 5). These elements are available from the De Lava1 Separator Co., Poughkecpsit,, X. Y. On the inner conical surface of the elements, a groove runs parallel to the circular opening. This is connected to outlets and inlets for steam. Into the flexible tube is fitted a hollow stainless steel connector plug (Figure 6). When the connector plug is pressed down into the funnellike receptacle by a screw cap, the groove 1748
is sealed by the surface of the tube, and steam can be passed t'hrough it. This can either be done continuously, when the tube is not in use, or intermittently if a material sensitive to heat is handled. (In a test with a very rich medium, meat extract,peptone-glucose broth supplemented with l O 9 j ascitic fluid, it was found t'hat put'ting the connection under steam pressui'e twice a day for 20 minutes mas enough to ensure sterility.) As it is very important that steam does not gain access i80 the interior of the apparatus, the seal along this edge of the groove is under direct pressure from the screw cap. If a very elastic tubing is used, instead of trhe simple connector plug, tho plug shown in Figure 1 is used, and the sealing along the othm edge is effected by the pressure from a, spring-loaded ring on t'he connector plug. If the screw cap is not d m v a t,ight, steam from the inside of the apparatus can escape through t,he steam outlet, which can be used to advant,age in some operatiorih. Actually, sterilization should be started r\ith hiscentral connection. Heat Exchanger. Because of the grorving importance of continuous sterilization of media and of rapid refrigeration of biological fluids, the design of the heat' exchanger (Figure 11) is
Figure 7.
Glass Wool Filter
Ddsigned to Fit Directly into Sterile Connection
Figure
9.
Rubber Figure 11.
Valve Used for Sterile Connection
Heat Exchanger
A.
Outlet with flexible tube connection E, C. Glass wool insulation D. Liquid pool E, 1. Liquid seal F, H. Steam coil G. Inlet with flexible tube connection
Figure 8.
Thermocouple Pocket Fitted with Sterile Connection
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L
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1
Figure 10.
Fermentor Arrangement in New Bacteriological Department of Karolinska lnstitutet A-F. G-J.
K,
1750
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Fermentors 1. Heat exchanger Storage tanks M. Hose pressure pump Stainless steel tubes fitted with flexible tube connections
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
Vol, 46,No. 9
ENGINEERING, DESIGN, AND PROCESS DEVELOPMENT significant. From the inlet, which is fitted with a sterile connection, the liquid is fed into the top portion of the apparatus, where it forms a pool. From the surface of this pool it runs down the inside of a number of cooled vertical stainless steel tubes and leaves the equipment by an outlet which is also fitted with a sterile connection. When no more liquid is passed through, the pool is automatically drained through a few small holes drilled through the vertical tubes just above their welded entry into the top portion of the heat exchanger. Cleaning of the straight tubes is an easy matter after the top and bottom lids have been removed. They are attached to the cylindrical part of the apparatus by means of a special trap which has been described (2).
Acknowledgment
The authors wish to acknowledge the assistance of Sven Warenius in making a number of test models of flexible tube couplings, and of Sven Goranson for valuable collaboration in designing the heat exchanger. literature Cited (1) H e d h , C.-G., Smed. Patent 145,828 (1954).
(2) HedBn, C.-G., Malmgren, B., Sundstrom, K. E., and Tornqvist, B., Acta Path. Microbial. Scand., 30, 284 (1952). (3) Phillips, C. R., and Kaye, S., Am. J . Hag., 50, 270 (1949). RECEIVEDfor review April 13, 1954.
ACCEPTED &fay 25, 1954.
Bactericidal Effects of Ultrasound Instrumentation and Techniques for Quantitative Studies LILLIAN A. RUSSELL
AND
ARTHUR M. BUSWELL
Illinois State W a t e r Survey, Urbana, Ill.
FRANCIS J. FRY
AND
ROBERT McL. WHITNEY
University of Illinois, Urbana, 111.
B
4 RICIDAL effects of ultrasound have been conclusively demonstrated by other workers in the field; however, in the equipment employed, quantitative control of the ultrasonic variables could not be obtained. A body of quantitative data based on the measurement and control of the physical variables of the ultrasonic field and sufficient to define the conditions most favorable to bacterial destruction would be of considerable advantage in evaluating this tool for commercial use and in outlining specifications for equipment to be used for municipal sterilization of xater and commercial sterilization of milk. The research described in this paper was initiated to supply this quantitative data. In the earlier studies a commercially available generator with quartz transducer was used on a pure culture of a single strain of Escherichza coli. More recent studies are being made on the same organism but uith the use of equipment designed for research purposes. This first section of the study is primarily concerned with describing instrumentation and techniques that are suitable for initial quantitative studies of the bactericidal effects of ultrasound.
Knowledge of Physical Phenomena of Ultrasonic Field Is Basic to Study
An experimental study that proposes to achieve the measure ment and control of the physical variables of the ultrasonic field, with the intention of correlating bacteriological effects with them, must be concerned first with an analysis of the characteristic phenomena of the system and the changes in the variables that produce them. Fry (6) reviewed these phenomena for biological systems in general and included in this category changes in temperature and in pressure, forces resulting from radiation pressure, and cavitation and its concomitants. Temperature changes in the absence of cavitation are of two kinds. A periodic temperature change arises from the adiabatic compression and expansion associated with a sound wave in a liquid medium. According t o Fry, this is of minor significance; i t is of the order of 0.01" C. for a sound-pressure amplitude of 10 atmospheres (an intensity of 35 watts per square em.) in water. September 1954
A gradual temperature increase is due to the progressive absorption of sound energy and its conversion into heat. For a plane traveling wave, the intensity decreases logarithmically with distance, and the amount of sound energy absorbed per unit volume of liquid per second a t any given distance from the crystal is expressed by the product of the coefficient of absorption and the intensity of sound a t that point. The magnitude of this coefficient increases with the viscosity of the medium and with the square of the sound frequency. Periodic pressure changes a t high frequencies are accompanied by a unidirectional radiation pressure. In relative magnitude much smaller than the alternating-pressure amplitude, radiation pressure manifests itself a t an acoustically reflecting interface with a force proportional to the intensity of the sound field. The alternating sound-pressure amplitude is proportional to the voltage applied a t the crystal and thus is a function of the square root of the intensity. A sound-pressure amplitude of 10 atmospheres is associated with a radiation pressure of 0.0024 atmosphere. At ultrasonic frequencies the rapid periodic recurrence of pressure changes becomes as important a factor as their absolute magnitude; for example, a sound-pressure amplitude of 10 atmospheres gives rise to a velocity amplitude of 70 om. per second, a particle amplitude of 0.11 micron, and an acceleration of 4 X IO8 cm. per second (40,000 g's). Unless measures are taken to prevent it, a body immersed in a liquid under such accelerations and velocity amplitudes becomes subject to cavitation, which occurs when the pressure on the surface of a body is reduced so low by the incapability of the flow to maintain contact with the body that a vacuum appears, or a t least a region saturated with the vapor of the liquid forms. The critical low pressure limit that can be reached before cavitation occurs is dependent upon the dissolved gas content of the liquid. Yumachi (14) found that with water saturated with air the limiting pressure was approximately equal to the vapor pressure of the liquid, but with even partially deaerated distilled water it was minus 1 atmosphere (absolute). Briggs, Johnson, and Mason ( 1 ) found that when liquids are degassed their natural cohesive power brcomes effective and they will withstand a
INDUSTRIAL AND ENGINEERING CHEMISTRY
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