US2163996A - Laboratory apparatus - Google Patents

Description

`une 27, 1939.

E. w.lFLosnoRF LABORATORY APgARATUs Filed Dec. 14, 19,'55 4 Sheets-Sheet l M6 INVENTOR 7) BYe ATTORNEYS June 27, .1939. Y E. w. FLosDoRF 2,163,995

LABORATORY APPARATUS Filed Dec. 14, 1955 I 4 Sheets-Sheet 2 INVENTOR` ATTO R N EYJ June 27, 1939. vla vw. Hoax-JORF- LABORATORY APPARATUS Filed Dec. 14, 1955 4 Sheets-Sheet 3 d .,J l w EN.. 2 3 M.

0 m, e 6 M Z 7 77 Il/ l w /M 6% l lNvENToR 7D BY gi a ATTORNEY:

.fm2-1,1939. A Ew. FLQSDRF 2,163,996

'LABORATORY APPARATUS Filed Dec. 14, 1935 4 SheetS-Sheefl 4 lNvEN-roR we /wz er 7) .BEY m gap, i

ATTO RNEYS other biologically active substances.

Patented June 27, 1939 UNITED STATES PATENToFFIcE LABORATORY APPARATUS Earl W. Flosdorf, Ardmore, Pa.,rassignor to The Trustees of they University of Pennsylvania, Philadelphia, Pa., a corporation of Pennsyl- Vania Appueetien December 14, 1935, serial No. 54,450

'7 Claims.

This invention relates to improvements in apparatus for the preservation of biologically active substances such as sera, protein solutions, bacterial cultures, viruses and other labile biological substances; and more particularly to improvements in apparatus for the treatment and preservation of biologically active substances by freezing the substance, dehydrating it from the frozen state under a high vacuum, and carrying out the operation in the final individual containers-in which the resulting product is to be kept until used.

In my companion applications, Serial Nos. 54,148, filed December 12, 1935, and 54,149, filed December 12, 1935, I have described such methods and apparatus for the preservation o'f such biologically active substances, and more particularly methods and apparatus for carrying outl such preservation on a larger scale. The present invention relates more particularly to miniature laboratory apparatus for the carrying outA of such processes and is intended primarily for research and experimental purposes where only a small' amount of the biological materials are to be preserved.

'I'he improved processes, set forth inrdetail in said companion applications, involve the treatment and preservation of the biologically active substances in the final container, with introduction of the substance in a liquid or semi-liquid state into the final container, freezing of the substance therein', dehydration of the substance from a frozen state and without melting, andwith continuation of the dehydration until the final dry product is produced with the use of a high vacuum, and sealing the container at the end of the process. The process is particularly valuable for preserving small unit or multiple unit amounts of the biological Isubstance in small individual containers in vwhich the final product is to be preserved and kept until it is to be restored for use. The process thus makes possible the preservation of small and clinically useful amounts or units, or multiple units, of serum and proved apparatus of the present invention enables such substances to be produced in the Y laboratory for research, experimental and similar purposes, on a small scale, and with similar advantages to those obtained in larger scale production according to the methods and apparatus of my said companionapplications.

The invention4 is particularly valuable for the production of so-called lyophile products by the rapid and complete freezing of the liquid The improduct at a low temperature, e. g. around -70 C. or lower, and with rapid vacuum evaporation from the frozen state and with self-regulation and automatic regulation of the temperature to keep the material frozen during the evaporation, even though exposed to room temperature.

The apparatus of the present invention com'- prises a manifold which is advantageously made integral with a main condenser, a secondary condenser attached to the main condenser by a rubber connection, a vacuum pump or other suitable vacuum producing means, small containers for the material to be preserved and suitable means for attaching the containers to the outlets of the manifold.

In one form of the'apparatus the manifold and main condenser are made of glass in the form of an integral unit, and with a separate, secondary glass condenser attached to the main condenser by a suitable rubber connection.

In another form of the apparatus the manifold and condensers-are made of metal, and the manifold and main condenser may likewise, and

with advantage, be made as an integral, unitaryI structure. v

The manifold of the apparatus, whetherof glass or of metal, has a, number of outlets of a shape and arrangement to receive a rubber stopper through which the exhaust tube of thein- ,Y

dividual containers extend, and with the outlets .of the manifold and the rubber Stoppers accurately proportioned to enable a vacuum-tight connection to be effectively-.and almost instantly obtained.

The main and auxiliary `condensers are constructed and arranged so that they may both be contained Within the same low temperature bath and the-main condenser is so constructed as to insure effective condensation of most of the water Amatic sublimation of ice from theupper part of the condenser to the lower part of the condenser is obtained during the carrying out of the process and as the level of' the refrigerating bath is lowas n ered, due to removal of refrigerant, e. g., solid carbon dioxide or Dry Ice.

The refrigerating mixture is advantageously one made up of Dry Ice or solid carbon dioxide suspended in a suitable organic liquid such as acetone, alcohol, ether, trichlorethylene, etc., but advantageously a bath containing the solid carbon dioxide and Methyl Cellosolve (the methyl ether of ethylene glycol). By surrounding the main and auxiliary condensers with such a bath, and by proper construction of the condensers, the water vapor removed from the frozen material, during the process, under the high vac uum maintained, is effectively condensed withom objectionable stoppage of the tubes through condensation of ice at parts of the apparatus where such condensation is undesirable.

It is important, in a small laboratory apparatus, where the parts of the apparatus are small, to have a construction and arrangement of the parts such that the process can be carried out in a reliable manner and with a minimum of attention and also with a minimum of danger of stoppage of the apparatus by condensation of ice in connecting tubes or pipes, While insuring effective condensation in the main condenser.

The individual containers for the biological material to be .treated and preserved may be small unit containers such as are described in my said companion applications, with suitable exhaust tubes, suitably proportioned, and with containers of proper shape and size, to enable the process to be effectively carried out; and the exhaust tubes of these containers are provided, in accordance with the present invention, with one-holed rubber Stoppers, accurately proportioned with reference to the openings in the manifolds, so that the stoppers can be quickly thrust into the tapered outlets of the manifold and secured therein with a vacuum-tight fit.

The vacuum pump or other vacuum producing means used should be such as will enable a high vacuum to be maintained, e. g. below 0.70 mm. mercury or deslrably-much below that pressure, e. g., within the range of from 0.01 to 0.05 mm. mercury. 'I'his range is easily attained with a suitable vacuum pump provided precautions are taken to have high vacuum-tight connections throughout the apparatus. To attain this result in a small laboratory apparatus it is important to have rubber tube and rubber stopper connections, where used, properly proportioned and arranged to accomplish this result.

The individual containers for the biological material may be of various types and can readily be standardized for laboratory and research purposes. For the preservation of sera and other protein solutions, etc., containers of 50, 25, 10, 5 and 2 ml. capacity have been standardized; containers of larger capacity can be used, but containers of the capacity mentioned are sufcient for the usual laboratory and experimental operations. Containers of 2 and 5 ml. capacity have been found convenient for preservation of materials in small amounts, e. g., virus suspension in amounts as small as 0.1 ml. The volume of material put into any container should ,not exceed about a half the capacity of the container. For amounts of materials in excess of about 1 ml. the containers are advantageously in the form of cylindrical glass containers in which the material can be frozen while the container is in a horizontal position, so as to give a proper ratio of surfaces and volume of the frozen material, as

aisance the manifold Fig. 2 shows an integral manifold and main condenser in elevation and with parts in section;

Fig. 3 is an end view of part of the apparatus of Fig. 2;

Fig. 4 is a separate view of the secondary condenser;

Fig. 5 is a view similar to Fig. 1 showing the apparatus made of metal;

Fig. 6 shows the separated manifold of Fig. 5 on somewhat larger scale;

Fig. '7 is an enlarged view showing the connection between the main and secondary condensers of Fig. 5;

Fig. 8 is a cross-section of a freezing trough with the freezing mixture and an individual container shown therein;

Fig. 9 is a plan of a freezing trough, also showing one individual container therein;

Figs. l to 15 show different .forms of containers with rubber Stoppers attached for connecting these containers with the outlets of the manifolds;

Fig. 16 shows one form of separated attachment, separatedfrom the container;

Fig. 17 ls a similar view showing a separated attachment of Fig. 16 as protected during sterilization and before attachment to the container; and

Figs. 18 to 2l show different forms of a sealed nal container with the preserved material therein.

The apparatus of Figs. 1 to 4 is a glass apparatus to be made, e. g. of Pyrex glass. The manifold l has a number of tapered outlets 2, which may vary in number in laboratory apparatus of varying slze,ve. g., from 8 to 24 outlets. The manifold is shown as integral with the main condenser 3.

The main condenser 3 and secondary condenser 4 are enclosed within the same container 5, shown as a double-walled container with a vacuum between the walls for heat insulation purposes. The container contains the freezing mixture, e. g., of Dry Ice and Methyl Cellosolve, surrounding the main and secondary condensers.

The connection between the main and secondary condenser is through a side outlet opening 6 having an accurately fitting rubber stopper 'i therein through which extends the inlet tube 8 of the secondary condenser.

The main condenser has an inlet tube extension 9 which extends down into the body of the condenser to a limited extent Vand terminates at the level of the top of the container 5 and the main condenser is supported in the container so that it extends to this level but does not extend further. a higher level. The arrangement of inlet and outlet tubes is such that the tendency to short circuiting of the vapors through the primary condenser to the secondary condenser is prevented The outlet tube 6 is arranged at to insure a vacuum-tight iit.

`serum or other biological material in the indiof the main condenser.

or minimized, and also such that the inner tubev extension 9 is protected from plugging by the deposit of ice therein. The main condenser has a tightly fitting stopcock I at its top with a side tube I I through which, and through the stopcock when in proper position, air may be admitted to destroy the vacuum at the end of the process.

The secondary condenser has its inlet tube I2 extending to near the bottom and has its side outlet I3 near its upper end. With effective operation in the main condenser, theamount of'.

water vapor carried over to the secondary condenser is so small that danger of plugging of the secondary condenser with ice is obviated. The rubber stopper connection between lthe main and secondary condensers, and the openingreceiving this stopper, are accurately proportioned The secondary condenser is connected through the side outlet I3 to a heavy rubber tubing I4 leading to the vacuum pump (not shown).

In the apparatus of Figs. 5, 6 and 7 a manifold I6, with its tapered outlets I7 and the main and secondary condensers I8 and I9 are made of metal, these condensers being enclosed Within a container 20, shown as a double Walled vacuum container, surrounded by insulation.

The manifold i6 has at one end an outlet connection 2I leading to the maincondenser, with which it may be integrally united b-y brazing or otherwise or with which it may be connected through a suitable vrubber tube connection shown at 22 which surrounds the lower end Vofthe manifold outlet 2l land also the tubular extension 23 at the top of the main condenser, as shown. rl'his tubular extension 23 which forms a part of the inlet to the main condenser extends down about I/z" to'l below the top of the condenser when this condenser is arranged as shown in Fig. 5. The condenser is suitably supported in the container 20 by supports 24 so that its top is about 1 below the top of the container 2li.

The main condenser has an outlet connection 25.

connected through the rubber tube 2li-to the inlet 27 of the secondary condenser.

The secondary condenser is of general U-shape and is arranged in the container2l] at one side Its outlet 28 is adapted for the connection of a rubber tubing 29 leading tothe vacuum pump (not shown).

The manifold of Fig. is shown as having an integral extension 3|! adapted to extend into a supporting pipe 3I carried by stand 32 for supporting the manifold.

The number of tapered outlets on the manifold of Figs. 5 to 7, as Well as the number on the glass manifold of Figs. 1 to` 4 can be somewhat varied to provide for the attachment of a larger or smaller number of individual containers.

A suitable freezing trough, for, freezing the vidual containers is shown in Figs. 8 and 9, the trough 33 having individual supports 34 for the individual bottles or containers, and a rocking device 35 by means of which if desired the conwhich also extends through the rubber' stopper 36.

The container 40 of Fig. l11 has a special rubber stopper 4I with an integral tubular extension 42 which is attached to one end of an L-shaped exhaust tube 43 which in turn extendsthrough the rubber stopper 36.

A clamping device of metal is shown yat 44 of adapted, when the process is completed, for sealing this tube by compression of this metal clamping device against the tube.

The container 45 of Fig. 12 is spherical in shape with a tubular extension 46 shown as connected through the tube 47 with the L-shaped glass exhaust tube 48.V The container 45 also has a side extension 49 with a perforable rubber stopper 59 closing the same With a vacuum-tight seal.

The container .5I of Fig. 13 is similar to that of Fig. 12, and the other parts are similar, except for the omission of the side extension 49 and stopper- 59. f

In Fig. 14 .the small container 52, which is of smaller size than the container of Figs. and 11, has a smaller rubber stopper 53 with integral tubular extension 54 having clamp 55 thereon similar to the clamp shown in Fig. 11.

The container 56 of Fig. 15 is of somewhat different shape and has the stopper 36 attached dlrectly to an integral tubular`extension of the container and has also another connection 51 with rubber stopper 58 therein.

Fig. 16 shows the rubber tube stopper, exhaust tube and manifold stopper of Fig. 1l separated from the container. These new rubber Stoppers, and nal container sealed by means of them, are claimed in my companion application Serial No. 106,105, led October 17', 1936. Fig. 17 shows the same or a similar assembly of container stopper, exhaust tube and manifold stopper with a protective bag 59 of paper or clot-h surrounding the stopper. This is used for sterilizing the assemblybefore use and protectsthe sterilized stopper until it is to be linserted in the container which is separately sterilized before the liquid material is placed therein. By sterilizing the container and the stopper and by moistening the stopper with sterile distilled water or dilute antiseptic solution when it is inserted, the material in the container is protected from contamination and asepsis is maintained.

The L-shape of the exhaust tube and the upwardly extending arrangement of the manifold outlets causes the exhaust tubes to have a downward direction when inserted`in `the manifold and this, together with the rapid oW-of vapor from the containers through the exhaust tubes, and the protection of the containers from ingress of air at the end of the process, by sealing them under their original vacuum, maintains `asepsis throughout the process and insures that the material originally placed in the container in a sterile condition will be protected from contamination, with certainty, up to the time it is restored for use. In Fig. 17 is also shown a cotton plug in the outlet and of the exhaust tube. This is left in the tube during the freezing and until 'the tube is to be attached to the manifold `and Fig. 11 surrounding the tubular extension 42 and Figs. 18 to 21 show the containers of Figs. 10, 11, 12 and 14 after the containers have been sealed, at the end of the process, the sealing of the containers of Figs. 19 and 21 being by heavy compression of the clamps M and 55 and by cutting olf the rubber tubes above the clamps; while the sealing of the containers of Figs. 18 and 20 is by fusing and drawing of the glass exhaust tube 39 of Fig'. 10 and of the integral tubular extension 46 of Fig. 12.

In the operation of the apparatus the serum or other biological material in unit or multiple unit amounts is introduced into the small individual containers, the containers are then closed and the rubber Stoppers or other connections added thereto to connect the containers with the exhaust tubes which are L-shaped and each of which is provided with a one-holed rubber stopper which may be quickly thrust into the tapered outlets of the manifold with a vacuum-tight connection. The material is then frozenand while in a frozen state is subjected to vacuum dehydration with automatic self-regulation of temperature and this operation is continued until the dehydration has been carried to the proper extent which is attained after the removal of ice is completed and the product is thereafter warmed up to around room temperature and kept at that temperature while the vacuum is maintained to insure completion of the dehydration.

The freezing of the material is advantageously carried out by rapid freezing to a low temperature such as by insertion of the small containers, with their liquid content of biological material in a bath of Dry Ice suspended in an organic liquid and by leaving the containers in this bath for a considerable time to insure thorough freezing and the establishment of equilibrium at a low temperature. This rapid freezing followed by proper rapid dehydration gives a valuable lyophile product.

The freezing is carried out, in the apparatus illustrated, by placing the cylindrical container on its side in the bath of Dry Ice and Methyl Cellosolve with the exhaust tube extending verticaily, as illustrated in Fig. 8. Care should be exercised not to freeze the serum or other material over the end of the exhaust opening. After solidification of the serum the containers are well covered with Dry Ice lumps and allowed to stand for about half an hour thus bringing them to a low temperature such that thawing will not occur during the attachment of the containers to the manifolds and during subsequent evacuation down to the working range at which evaporation automatically keeps the material frozen, e. g., at a vacuum below about 0.7 mm. mercury. Not even the surface of the serum should be allowed to melt or otherwise frothing will occur. For processing the material in an effective and reliable manner, the size and shape of the cylindrical containers are advantageously such that the layer of frozen material on the side while in a horizontal position therein is not less than 3 millimeters in thickness at the vertical diameter nor more than J5 millimeters, and also such, as already pointed out, that the volume of the frozen material does not exceed about one-half the volume of the containers. When amounts on the order of 25 m1. in a 50 ml. container are frozen, the container should be rocked somewhat during freezing so that a trough will form in the center, thereby reducing the thickness and increasing the evaporation area.

In the operation of the apparatus, Dry Ice or of the main condenser.

solid carbon dioxide broken into' lumps approximately 1/2" in diameter is placed in the container around the condensers up to the mouth of the container. The organic liquid, e. g., Methyl Cellosolve, is then poured in slowly to Within a short distance of the top of the container in Fig. l, e. g., about 2" below the top in'the apparatus shown where the main condenser has an outside diameter of about 65 mm. In the apparatus of Fig. 5 the organic liquid is filled into the container to within about V4" or 1/2" of the flat top The Dry Ice consumed in chilling the condensers at the outset of the process is replenished but no further additions are made until near the end of the process, e. g.. for about eighteen hours.

After each container is removed from the freezing bath, the cotton plug, shown in Fig. 8, is removed from the end of the exhaust tube, the special manifold stopper on the top is lubricated with sterile or distilled water or dilute antiseptic solution and the stopper is then quickly thrust with a twisting motion deep into one of the outlets on the manifold. This requires only two or three seconds per container. Rapid attachment is essential in order to avoid surface thawing especially when the containers have amounts less than about 5 ml. in them. When all the manifold outlets are thus filled the vacuum pump is started immediately. If some of the outlets are not connected with containers, they'should be plugged with a solid rubber stopper with a vacuum-tight fit. Within a short time the outsides of the containers usually begin to frost, due to condensation and freezing of atmospheric moisture. From this point on the process proceeds automatically with no need for further attention until the Dry Ice is replenished after about eighteen hours. As dehydration nears completion and the evaporation rate diminishes, the temperature of the containers slowly rises to that of the room and the frost thaws. The process is continued for some time thereafter to insure completion of the dehydration while the material is at a high temperature, and with a high differential between the vapor tension of the water in the material at the high temperature and the low vapor tension in the condenser.

In freezing the material in containers having small amounts, less than 1.0 ml. per container, as in the case of bacterial vaccine, for example, freezing of the container on its side is unnecessary in order to secure a large evaporating surface and with such small containers it is advantageous to have the freezing pans in such a position that the material can be frozen after attachment of the containers to the manifold but prior to evacuation. With small single containers containing such a small amount of material, it is of advantage to keep the containers in the freezing mixture until the proper degree of evacuation has been obtained and the automatic temperature control is secured. 'I'he process is thereafter carried to completion with automatic temperature control, as hereinbefore described. With larger containers containing a somewhat larger amount of serum or other material, and with the ratio of volume and surface hereinbefore described, and in containers of proper size and shape, the containers can be rapidly attached, after removal from the freezing bath, to the manifold, and the manifold rapidly evacuated to start self-refrigeration or automatic temperature control.

At the outset of the process most of the frozen condensate collects near the top of the main condenser but as the bath level around the condenser falls, due to consumption of the Dry Ice in condensing the vapor, the condensate sublimes downwardly in the condenser. In this way the entire vbottom portion of the condenser is lled. Replenishing of the Dry Ice is undesirable during this stage of the process as it-would interfere with or prevent this resublimation in the condenser. As the level of 'the solid-liquid mixture in the cold bath around` the condensers becomes lower, the cold and heavy carbon dioxide gaseous atmosphere above the mixture will still extend to the top of the container and consequently to the level of the bottom of the inlet tube in the main condenser. This carbon dioxide atmosphere is of sufficiently low temperature to cause preliminary freezing in the main condenser of the vapor coming over and to prevent short circuiting of the vapor over into the secondary condenser. The condensate consequently assumes a shape and position such as illustrated in Fig. l. Ordinarily the condensation in the main condenser is sufficiently complete so that only about 1% of the total condensate collects in the secondary condenser. In order to facilitate the removal of the last traces of moisture from the substances being dehydrated it is advisable to replenish the Dry Ice in the container around the condenser near the end of the process, e. g., after about eighteen hours, somewhat more or less, depending somewhat upon the size of the apparatus and the operation of the process.

By following the procedure described, as much as 99.96% of the original water content of serum may be removed, giving a product containing only about 0.5% of moisture in the final product as determined by desiccation in an oven at 110 C. The time required to obtain this degree of desiccation depends on the quantity of serum or other material to be dried. Amounts of the order of 0.1 ml. per container are dry within a few hours; while amounts of serum, etc., up to 25 ml. can be dried in around eighteen hours, although it is safer to allow a longer period, e. g., of about twenty-two hours. This enables the apparatus to be kept operating on a daily schedule of twenty-two hours process time, allowing two hours for sealing off one set of containers and preparing the containers for the next operation. The last step of the process is that of sealing olf the containers without breaking the vacuum. If glass exhaust tubes are used the seal is m'ade with a suitable oxygen torch fitted with a double tip. By careful, slow and even heating of the tube on all sides at once, while pulling on the glass, an excellent seal of Pyrex exhaust tubes up to 5 mm. internal diameter is readily obtained, giving a sealed tube such as illustrated in Figs. 18 and 2o. p

When using the rubber tube stopper, with an integral rubber tube extending from the stopper,

as shown in Figs. 11 and 14, the clamping sleeve, e. g. of annealed brass of suitable thickness, may be squeezed rmly with pliers that close with ilat and parallel jaw surfaces and with a compound The vacuum is maintained throughout `the` process and during the sealing operation. The vacuum is now turned oil and air is admitted to the apparatus by means of the glass stopcock at f the top of the main condenser in the apparatus of Fig. l or by withdrawing one of the manifold Stoppers from a manifold outlet in the apparatus of either Fig. 1 or Fig. 5. Where glass exhaust tubes are used and are sealed with a flame the vacuum in the apparatus is not destroyed by the sealing of the tube. With rubber exhaust tubes the tube is sealed while the vacuum is maintained additional runscan be carried out piovided the condenser capacity is adequate; If the condensers have insufficient capacity remainingfor the next run they must be emptied. This requires melting of the ice in the condensers and the removal of the water from the condensers before the next run is begun. Melting of the ice can be'accomplished. by surrounding the condensers with warm water at 30 to 50 C. and by then siphoning out the condensate in the condenser when it has thawed, which can be vaccomplished by removal of the stopcock plug in the apparatus of Eig. l or by disconnecting one oi the outlets of the main condenser in Fig. 5 and introducing a rubber Siphon tube. The secondary condenser is easily disconnected to permit pouring out of any condensate. Once the condensate has thawed the condensers should not be packed with dry ice-until they have been emptied. Otherwise the freezing of the condensate in the condensers would break or injure the condensers due to expansion of the water on freezing.

-In apparatus of Fig. 1 it willbe noted that the main condenser extends somewhat above the top of the surrounding. container and that the inlet tube extends down to4 about-the level of the top of the container While the outlet tube is at a higher level. In the metal apparatusof Fig.

4 .the top of the main condenser is somewhat below the top of the surrounding container but the level of surrounding liquid at the outset is a little below the flat top of the main condenser so that the top ofthe main condenser is surrounded by an atmosphere of carbon dioxide. The arrangement in both cases is such as promotes rapid and effective condensation in the main condenser with a minimum of danger of plugging of the connecting pipes with the condensed ice.

In order to empty the condensers, the secondary condenser of Fig. 1 can be readily detached from the main condenser because of the rubber stopper connection; and the secondary condenser of` Fig. 5 can similarly be disconnected by disconnecting the rubber'tube-Which connects them.

lifter emptying the condensers, and lbefore .starting up 'the apparatus, all oi the rubber connections on the `condensers should be made before the condensers are chilled and they should not be disturbed while they are cooling, otherwise small pieces of icecondensed from the atmosphere tend to collectvon the metal around the frozen rubber parts and cause leakage.

The laboratory apparatus described, and. the method carried outwith it, are adapted for use for a wide variety of purposes for the treatment Vand preservation of biological.substances on a.

small scale such as for laboratory, research and control purposes.

'I'he final containers, sealed while under a high vacuum, and without destroying the vacuum under which the dehydration was carried out, will contain ythe products preserved without danger of contamination from air or moisture, or from micro-organisms or other contaminants. It is possible to insure asepsis through the process so that the material finally sealed in the container in a dehydrated state has `with certainty been protected from contamination after it was placed in the container and frozen,

The nal product can be restored without destroying the vacuum, where a perforable rubber closure is used, as in Figs. 10, 1l, 12, 14, 15 and 18 to 21. The rubber Stoppers oi Figs. 10, 11,- 12, 15, 18, 19 and 20 have thin portions for the introduction cf a hypodermic needle to admit liquid without destroying the Vacuum. With the integral rubber tube stopper of Figs. 14 and 21 the rubber tube itself can be perforated with a hypodermic needle, etc., to admit water i'or restoration; and the rubber tubes of Fig. 19 can similarly be perforated instead of the short, separate, thin portion. The rubber stopper shown in Fig. 8 and also in various other figures has an opening extending part way through the stopper leaving a thin portion where the stopper can be readily perforated by a hypodermic needle.

The introduction oi' water or normal saline solution to restore the product, without destroying the vacuum, facilitates redissolving of the solid product since the product can be thoroughly wet with the water while the vacuum is maintained,

. and then, when atmospheric pressure is admitted,

the pressure insures that the water is forced into al1 parts of the dry material and this thorough penetration of the water is not interfered with by contained gas; whereas, ii' the vacuum had been destroyed before the water was introduced, penetration would be retarded by the gas contained in the pores of the solid material.

The following table shows typical sizes of containers for use with the apparatus and process, with an indication of the approximate container volume, the maximum volume of serum or other material to be processed and to be contained in the container, the body length, diameter and wall thickness, and the length and diameter ofthe necks of the containers to adapt them for the rubber stopper closures by which an effective vacuum is to be maintained.

Neck inside Approxi- Maxi- Body Wa diameter mate conmum Bod outside thick Neck tainer serum ngt diameness length volume volume ter Large Small end end Ml. Ml. Mm. Mm. Mm. Mm. Mm. Mm.

50 185 35 2. 0 15 15. 2 13. 7 50 25 110 28 1. 5 16 l5. 2 13. 7 25 13 80 22 1.6 15 15.2 i3. 7 12 (i0l 20 1. 2 16 l5. 2 13. 7 d 3 45 l5. 5 1. 0 15 15. 2 13. 7 2 1 Sphere 22 l. 0 10 5. 4 4. 5 2 1 22 16 1. 0 1l 7. 8 6. 8

and the exterior surface in contact with the walls oi' the container, as more fully described in my said companion applications.

Suitable rubber stoppers for use with the containers of the above table and for use in attaching the containers to the manifold and also 'or connecting the secondary condenser with the main condenser are shown in the following table:

The stopper No. 1 of the above table is illustrated in Figs. 8, 10 and 18. The rubber tube stopper of, No. 2 is illustrated in Figs. ll, 16 and 19. The small rubber tube stopper No. 3 is shown in Figs. 14 and 21. Stopper No. 4 is stopper i of Fig. 1 for connecting the secondary condenser with the main condenser. The manifold stopper No. 5 is indicated at 36 in the drawings.

For high vacuum tightness, such as the maintenance of a high vacuum around 0.01 to 0.05 mm. during the process and when the final container issealed, a great compression of the rubber of the rubber stopper is essential. This is obtained by tapering both the neck of the container and the stopper and by lubricating the stopper before it is inserted so that there is a maximum of compression on the entire length of the neck surface. A similar tight fit is obtained between the main and the secondary condenser and between the exhaust tubes and the manifold outlets to which the stoppers are attached.

In general the amount of material introduced into the individual containers will be around onehalf or somewhat less of the volume of the container. The final dehydrated or lyophile product will have a volume approximately that oi the original liquid or frozen material although in weight it: will represent only a small fraction of the weight of the original liquid. 'Ihe facility with which a small amount of a lyophilic product can be quickly restored to a liquid state with properties comparable with those of the original j liquid material before treatment makes the present process and apparatus a valuable one for use on a small scale for experimental and laboratory purposes and the treating of a small number of multiple unit amounts of biological materials to preserve them for long periods of time such that they are available for clinical or other purposes.

The apparatus has proved well adapted for use in a number of laboratories for the preservation of sera of various kinds, such as a variety of normal and immune sera, viruses, Aenrymes, various proteins, bacterial cultures, human milk, etc.

In this application I claim the new apparatus herein described for use in the dehydration of biologicals and the like. The new process is claimed in my divisional application Serial No. 126,057, filed February 16, 1937.

I claim:

1. An apparatus for the dehydration of biological substances in a frozen state in small individual containers comprising a manifold having a number of upwardly and outwardly extending outlets, each adapted for receiving witha vacnum-tight fit a rubber stopper attached to the exhaust tube of a container of the frozen material, a main condenser directly connected to the manifold, .a secondary 4condenser connected' to the first condenser and in turn adapted to be connected to a vacuum pump, a container for containing both the main condenser and the secondary condenser with space therein for surrounding the condensers with a mixture of Dry Ice andorganic solvent, the connection between the main condenser and secondary condenser being above the top of the container, and the inlet to the main condenser extending downwardly a short distance into the main condenser and terminating in a downward direction at a level lower than the outlet from the main condenser to the secondary condenser.

2. An apparatus for the dehydration of biological substances in a frozen state in individual containers under a high vacuum comprising a manifold having a number of upwardly and outwardly extending openings, each adapted to receive with a vacuum-'tight fit the rubber stopper of the exhaust tube of an individual container, a number of individual containers for the frozen material each with an L-shaped exhaust tube, one end of which extends downwardly through the rubber stopper into the opening in the manifold, when the container is attached thereto, a condenser directly connected with the manifold, an insulated container surrounding the con-l denser and adapted to contain the mixture of Dry Ice and organic liquidtherein around the condenser, said condenser having an inlet opening extending downwardly a short distance into the condenser and directing the vapors downwardly therefrom into. the condenser. and said condenser also having an outlet arranged at a higher level leading to a vacuum producing means.

3. An apparatus such as set forth in claim 1 in `which the main condenser and manifold are of glass and are made integral with each other, and in which the top of the main condenseris arranged above the top of the container, and the inlet tube to the main condenser extends'down to a point about even with the top of the container surrounding the condenser.4

4. An apparatus such as set forth in claim 1 in which the manifold and condensers are of metal, with the manifold directly connected to the top 0f the main condenser and having a discharge tube extending downwardly a short distance into the main condenser and in which the secondary condenser is of general U-shape and connected with the main condenser through a rubber tube connection. Y

5. An apparatus such as set forth in claim 1 in which the connection between the main condenser and secondary condenser is located above the top of the surrounding container and in which the inlet to the main condenser extends down into the main condenser and terminates at a location approximating the top of the surrounding container.

6. An apparatus such as set forth in claim 1 in which the secondary condenser has an inlet at the top and a central tube extending downwardly to near the bottom of the secondary condenser and in which both the inlet and outlet of the secondary condenser are arranged above the top of the surrounding container.

7. An apparatus such as set forth in claim 1 in which both the main and secondary condensers are made of glass and secured together through a rubber stopper connection with a vacuum-tight

Images