Localization of Certain Chemical Constituents in Plant and Animal Tissues An Electrographic Method HERMAN YAGODA, 540 West 123rd St., New York, K. Y.
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HE electrographic methods for the analysis of polished surfaces of minerals and alloys described b y Jirkovsky ( 3 ) and Glazunov and Kiivohlavy ( 2 ) suggest a n important application in the study of the distribution of the inorganic constituents within a section of tissue. I n early experiments conducted by the writer, thin sections of tissue were incinerated on glass slides plated with a film of platinum and the electrically contacted ash was connected in circuit opposite a sheet of platinum] the two electrodes being separated by a reagent paper moistened with a n electrolyte. Crude patterns were thereby obtained t h a t revealed the presence of iron and copper in the ash of liver tissue. While this laborious ashing and mounting technique provides a specimen simulating a polished mineral surface, its electrical conductivity is poor and the ash skeleton is frequently distorted b y contact with the moist reagent paper. It occurred t o the writer that the electrographic technique might be applied more successfully b y electrolyzing a thick cut of the original tissue. This limits the method to constituents that are present in a n ionic condition, and to specimens of fair rigidity. Electrographs of freshly cut vegetable sections on sensitized adsorbent papers showed that the method could be used for rendering macrographic patterns revealing the presence of the chloride] sodium, and potassium ions. I n order to make the technique serviceable in the study of fine detail, a structureless matrix for the reagent was necessary, as the fibrous composition of absorbent papers interfered with the microscopic examination of the electrographic print. After considerable experimentation thin plane castings of plaster of Paris were substituted for the filter papers commonly employed in the technique. It was also found possible t o obtain electrographs of high clarity through the use of suitably sensitized gelatin-coated papers. The plaster sheets are prepared b y pouring a thin slurry consisting of three parts of plaster and two parts of air-free mater into a suitable mold and allowing the sheet to set. The casting is also sensitized during its preparation by incorporating into the mix water insoluble reagents such as zinc sulfide, barium carbonate, etc. More detailed procedures for the preparation of thin plaster sheets will be published in a separate article describing their application in capillary and chromatographic analysis. The present article is limited to a detailed description of the method employed by the writer for the execution of chloride patterns exhibiting the cellular detail of vegetable tissues through the use of paper coated with a gelatin emulsion of silver chromate. The use of the sparingly soluble silver chromate as a fixed source of silver ion in the gelatin provides a highly sensitive emulsion that is specific for the halogen ions, and in view of the minor occurrence of bromides and iodides in normal tissue the ultimate pattern obtained after the emulsion has been properly processed may justifiably be termed a chloride pattern.
paper, single weight, Eastman Kodak Company, Rochester. N. Y.) with a solution of potassium chromate, blotting off excess fluid, and immersing the paper rapidly in a bath of silver nitrate. The quality of the sensitized emulsion varies with the concentrations and temperatures of the chromate and silver-ion solutions, the order of their application to the gelatin, and the time the sheet is kept in the baths. After considerable trial the following procedure was finally adopted : The imbibition paper is cut into sections to fit the upper electrode of the electrographic apparatus and each piece is immersed individually in a 2 per cent solution of potassium chromate for exactly 2 minutes. The paper is drained, and blotted between sheets of lintless absorbent paper similar to C. S. & S. No. 211 until the gelatin is completely freed from superficial chromate solution. The sheet is placed for 10 seconds in a 1 per cent solution of silver nitrate contained in a vessel of sufficient capacity to permit the complete immersion of the paper in a vertical position. The paper is then blotted and washed free from silver nitrate by passing it through a series of three trays containing d ~ s tilled water, blotting the surface a t each change of bath. Emulsions of almost identical silver chromate content result if the temperature of the reacting solutions is kept a t approximately 25' C. If the sensitized papers are to be used the same day, the wet paper is placed in a saturated solution of calcium sulfate. Otherwise, the papers are dried a t room temperature and kept in an air-tight container. The dried papers must be soaked for a t least 30 minutes in a saturated solution of calcium sulfate in order to render the dense backing electrically conducting.
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FIGURE1.
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ELECTROGRaPHIC
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10cm
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APPARATUS
a. Removable crossbar
b. Setscrew
Sensitization of Gelatin Papers with Silver Chromate
c. Steel pins
d . Aluminum electrodes e. Absorbent mats a b o u t 1 m m . thick (C. S. &
f. Sheet of sensitized emulsion or plaster
A uniform emulsion of silver chromate is obtained by moistening the gelatin film of a gelatin-coated paper (imbibition
S. No. 470)
Sponge rubber support a b o u t 15 m m . thick h . Hardwood base 2. Bakelite angles supporting m a t e and emulsion Q.
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NOVEMBER 1 5 , 1940
ANALYTICAL EDITION
The freshly prepared silver chromate emulsion has a bright red color which darkens on exposure to direct sunlight. The photosensitivity of the emulsion is of no moment, as excess reagent is removed during the processing of the electrographic print. The sensitivity of the emulsion to halogen ions necessitates great care in the handling of the paper. The moisture film carried b y the skin contains sufficient sodium chloride to leave finger prints on the processed sheet. T h e emulsion prepared according to this procedure contains approximately 120 micrograms of silver chromate per sq. cm., a quantity equivalent stoichiometrically to about 25 micrograms of chloride ion. The sensitivity of the gelatin test paper was determined by eraporating to dryness 0.008-cc. droplets containing known quantities of sodium chloride and reducing the resultant spots of silver chloride to black metallic silver, following the identical procedure utilized in the development of the chloride pattern. This showed that 0.0008 microgram of chloride ion at a concentration limit of 1 to 10,000,000 could be readily differentiated from a similar spot (area 10 sq. mm.) produced by a droplet of distilled water. The white stain of silver chloride on the red background of the emulsion can be observed only a t chloride-ion concentrations exceeding 1 to 20,000. This concent'rabion limit is of the same magnitude as the one reported b y Clarke a n d Hermance ( I ) , using absorbent papers impregnated with the same reagent. As a n excellent print is obtained a t concentrations of 1 t o 500,000 after reduction of the silver chloride, the electrically exposed emulsion should always be developed even when a white image is not, apparent on the surf ace.
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upper electrode is then fitted with a moist mat and a sheet of the silver chromate paper that has been thoroughly wetted with the electrolyte. Excess liquid should be removed from the emulsion by brushing the surface with a lintless absorbent paper before it ia finally clamped in position. The two electrodes are connected to appropriate poles of a 45volt battery in series with a 500-ohm variable resistance and a meter having a range of about 100 milliamperes. The emulsion is brought in contact with the section, and the potential is applied and maintained until about 0.2 coulomb of electric charge per sq. cm. has passed through the surface of the tissue.
FIGURE 2 . ENLARGEMENT PRINT OF CHLORIDE PATTERN OF CELERY SPECIMEK 11, X 20
Preparation of the Specimen To secure uniform electrical contact with the emulsion, the specimen must be provided with two plane and parallel surfaces. Plants whose cross-sectional area does not exceed about 4 sq. cm. are easily faced with the aid of a guillotine assembled from two razor blades that are free from bodily cutouts (Schick Magazine Razor Company, Kew York) mounted in a U-shaped frame, separated from each other by a distance equal to the desired thickness of the section. The thickness of the slice can be varied from a minimum of about 1 mm. in accordance with the internal resistance and the rigidity of individual specimens. An average thickness of about 3 mm. was found suitable for the majority of the household fruit and vegetables studied by the writer. Complete sections of larger specimens can be cut by means of a long knife guided by properly spaced slots in a miter box. Nonrigid animal tissue, in the absence of a freezing microtome, is best cut using a single unguided razor blade. The surface to be electrographed is placed in contact with a lintless absorbent paper, shifting the specimen until the face is freed from exuding fluids. Fish and animal tissue tends to adhere to paper and the surfaces of such specimens are best drained by means of plane porous castings of plaster of Paris. These are prepared by mixing 35 grams of plaster with an equal volume of water and pouring the thin slurry into a square brass frame of 10-cm. edge and 3 mm. deep supported on a glass plate.
Electrographic Procedure The apparatus illustrated in Figure 1 has been designed with the object of reducing to a minimum the period of mechanical contact of the specimen with the emulsion. The unit consists essentially of two aluminum electrodes. The lower one is supported on a layer of sponge rubber. The upper one, carryin the emulsion, slides freely in a removable crossbar. Thick absor%ent papers moistened with a saturated solution of calcium sulfate are placed on the metal electrodes to facilitate uniform electrical contact with the specimen. The section is placed on the lower electrode with the drained surface uppermost. Some specimens, particularly of fish and animal tissue, tend to cling t o the emulsion a t the completion of the electrolysis; this can be avoided by providing the edges of the electrode with thin strips of perforated Bakelite, passing two lacquered steel pins through the perforations penetrating into opposite sides of the specimen for a distance of 1 to 2 mm. The
The electrical exposure for prints of optimum detail is governed b y the quantity of ionic chloride, and may vary with different specimens from a few seconds t o about 2 minutes. It is not possible to formulate proper periods of exposure, owing to the variation in the chloride content of different specimens from the same species. The data presented in Table I are indicative of the range of exposures that resulted in prints of sufficient clarity for microscopic study. The electrically exposed emulsion is treated with 5 per cent nitric acid a t approximately 25" for about 1 minute, causing the complete decomposition of the red silver chromate. The print is washed in distilled water for a t least 15 minutes until it is freed from acid and silver nitrate. The paper is then placed in a bath of Eastman print developer, formula D-72, diluted with two volumes of water until the silver chloride is reduced to metallic silver. The exact developing time is best gaged from the appearance of the print. It is convenient to identify different prints by writing on the edges of the moist silver chromate emulsion with a sharp-pointed fragment of fused potassium chloride. I n the developing bath the silver chloride inscriptions are the first markings that become visible, and the paper is rotated in the bath until the notations acquire a jet-black color. At this point the electrographic image is usually a t its greatest clarity and further developing action is terminated by immersing the print in 1 per cent acetic acid for a few seconds. The paper is finally washed in running tap water for about 30 minutes. The dry prints are mounted on rigid cardboards with the aid of rubber cement. The chloride pattern is best examined for fine detail with a metallographic microscope or similar instrument provided vith a vertical illuminator. Sufficient time has not elapsed to enable the writer to make definite statements concerning the stability of the pattern on aging, b u t i t would appear that properly washed prints should approximate the stability of pictures on photographic paper. It is good practice to make a phot!ographic copy of the pattern, as enlargement prints made from the negative facilitate the study of macro segregation. Typical enlargement prints of the chloride patterns of sections of celery and carrot are reproduced in Figures 2 and 3.
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Metallic sulfides produce black images of silver sulfide when mineral specimens containing galena or pyrites are electroI n the absence of ionic bromides and iodides the resulting graphed on silver chromate emulsions. These markings do black markings on the processed Print can be interpreted as not dissolve completely in cold 5 per cent nitric acid. The mirror images of the location of ionic chloride in the tissue. presence of sulfide ion in n o m a 1 tissue is highly improbable, and if found would reveal itself by black markings on the unprocessed print. TABLE I. CHLORIDE PATTERXS O F VEGETABLE TISSUE T h a t the image is formed bv chloride ions migrating from points within the tissue to the anode and Specimen Area Current Time is not merely a contact print of Sq. Cm. Mallramp. Sec. the film of dissolved salts residing I. Celery on the drained surface of the speci(near root) a 0.6 0 30 men is demonstrated by the series b 0 30 of prints in Table I of celery speciC 3 5 30 d 3 5 15 men I. I n this reproduction (enlarged threefold) a and b are two a b C successive contacts of the specimen with the sensitized emulsion and c and d are electrographic prints produced by the passage of 0.1 and 0.05 coulomb electric 11. Celery (middle region) 1.5 6.0 30 charge, respectively. It is evident that electrograph c is of greater intensity and clarity than the initial contact prints. Careful inspection of the four images also shoivs the presence of specific markings in the eIectrographs which are entirely lacking in the contact prints, particularly the pronounced circular fibers on the 111. P o t a t o outer periphery of the section. (full section) 14.5 1s 00 I n view of the difficulty of securing perfect electrical contact with the tissue, white regions within the chloride print of a homogeneous specimen do not prove the absence of this element, as a n external resistance may have been superimposed as a result of the localized inclusion of air between the tissue and the emulsion. Such voids in the IV. P o t a t o (partial section) 5.7 22 30 pattern can be distinguished from true segregations in that they are not reproducible in successive prints. The number of exposures that can be made from a given sample depends principally on the abundance of the chloride ion. I n the study of one potato section, six successive prints were made using V. Cauliflower (green stalk) 1.4 10 30 a current density of 3 milliamperes per sq. cm. and a n exposure period of 1 minute. All six patterns were identical, the last print being of about the same intensity as the initial electrograph. I n some carrot specimens, however, the chloride ion appears to be completely depleted after the third exV I . Carrot 7.1 Pli GO posure. The question also arises whether the reproducible segregations are a measure of the abundance of the chloride ion, or merely represent differences in the electrical conduc-
Discussion of Results
ANALYTICAL EDITIOK
NOVEMBER 15,1940
FIGURE 3.
ESLARGEhIENT P R I N T O F CHLORIDE PATTERN O F CARROT SPECIMEN VI, 10
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in question. Such analyses were made on the carrot section shown in Figure 3, removing annular layers about 1 mm. thick over a length of 3 cm. from the inner core and the surrounding wall as indicated by arrows a and b, respectively. Analysis of the aqueous extracts from these samples revealed a chloride content of 0.13 and 0.08 per cent, which agrees in magnitude with the relative intensities of the tm-o areas on the print. The interpretation of the chloride pattern is further complicated b y the intensification of the print along the outer borders of the section. Thus, i t would appear from inspection of the complete section of the potato (Table I, specimen 111) that a considerable segregation of chloride occurred in the skin. However, the electrograph of a partial section (Table I, specimen IV) shows an intensification along the three cut edges similar to that of the skin itself. This edge effect is probably caused by the more intimate electrical contact between specimen and the emulsion along the interface. An analogous condition may exist on a n ultramicro scale along the wall of individual cells of the potato, as indicated by the microphotograph of its chloride pattern in Figure 4. With the present rudimentary development of the electrographic technique the chloride pattern can be interpreted only as a qualitative pictorial rendition of the surface of a section proving the presence, but not necessarily the absence, of the chloride ion in different parts of the tissue. The clarity of the chloride pattern is dependent on the plane character of the cut surface, its rigidity, and the absence of liquid seepage from the specimen while i t is being electrographed. Compact vegetable tissues yield electrographs of sufficient clarity to permit microscopic resolution of cellular detail. Typical structures are exhibited, under varying degrees of magnification, in Figures 4, 5 , and 6. These figures are presented only as examples of the definition obtainable by the technique described. They should not be interpreted as definitive of the chloride disposition within the respective species. Fresh garden specimens were not available for study and the electrographs undoubtedly contain abnormal features owing to the state of decay of the samples and to the injury of certain cells during the sectioning. The extraordinary definition and delicacy of the method are characterized by the photomicrograph (Figure 5 ) of a
PHOTOMICROGRhPHIC DETAILFRO?d CHLORIDE P.4TTERX O F POTATO, 100
FIGURE 4.
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tivity of the respective regions. A general solution to this problem is a matter of considerable difficulty, as the conductivity of the tissue is itself proportionate to the concentration and character of the ions present in the cellular fluids, and a simple measurement of the resistivity of various points of the tissue would not suffice even if experimentally feasible. In the case of macro segregations occurring over areas sufficiently large to provide ample samples for microchemical analysis, the validity of the electrographic pattern can be tested by measuring the chloride content of the aqueous extracts from the areas
FIGURE5 . PHOTOMICROGRAPHIC DETAILFROM CHLORIDE PATTERK OF CAULIFLOWER, X 500
INDUSTRIAL AND ENGINEERING CHEMISTRY
FIGGRE 6.
PHOTOlIICROGRhPHIC
DET.ULFROU
detail from the chloride pattern of the cauliflower specimen V shown in Table I. The capillary unit indicated b y the arrow on Figure 5 corresponds to a n area of 1.2 x 10-5 sq. cm. on the original chloride pattern. By comparing the intensity of the region with spots produced by droplets of known chloride content i t is estimated that about 2 x IO-'* gram of chloride ion reacted with the emulsion in the formation of the image of this capillary. The photomicrographs also suggest that the electrographic process can be employed in the estimation of cell dimensions. Chloride patterns of animal tissue are not of comparable clarity with those obtained from plant sections, owing to the mechanical difficulties of securing a properly faced section. After electrographic contact with animal tissue, gelatinous matter usually adheres to the emulsion. This can be removed while the sheet is in the nitric acid bath by brushing the surface with a soft brush. Since the electrographic method requires the use of freshly sectioned tissue, the writer has not been able to make detailed application of the technique on animal tissue. I n one experiment, made possible through the kind collaboration of W. H. Summerson, approximately plane sections cut from the liver, heart, and brain of a large white r a t immediately after the death of the animal mere electrographed on a silver chromate emulsion. hIicroscopic examination of the resultant chloride patterns revealed typical structures characteristic of the respective organs. A detail from the brain, showing the skin layers, is reproduced in Figure 7.
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CHLORIDE P A T T E R S O F CARROT,
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Potassium patterns can likewise be obtained by moistening the plaster sheet with a freshly prepared 20 per cent solution of sodium cobaltinitrite. After electrographic exposure the sheet is permitted to dry and excess reagent is removed by repeated extractions with 1 per cent acetic acid. The yellow outlines of potassium cobaltinitrite residing on the plaster can be rendered more suitable for microscopic examination by exposing the surface t o hydrogen sulfide vapors, which produce a superficial film of black cobalt sulfide on the yellow pattern. In the case of the potato, the ash of which is very rich in potassium, an exposure of 15 seconds at a current density of about 15 milliamperes per sq. em. suffices for the rendition of a potassium pattern which approximates the corresponding chloride pattern in its exhibition of macro detail.
FIGURE 7. DETAILFROM CHLORIDE P.4TTERS OF SECTION O F RAT BRAIN, X 12.5
Electrographic Patterns of Alkali Metals L4mongthe other ionic constituents present in normal tissue
of which electrographs can be made are sodium and potassium. Unfortunately, distinctive color reactions are not available for either of these elements that are comparable in sensitivity with the silver chromate emulsion employed in the formation of the chloride pattern. Electrographs of fairly sharp definition revealing the presence of the common alkalies in sections of potato, celery, radish, and carrot have been obtained, using as the recording medium sheets of plaster of Paris 1 mm. thick moistened with appropriate reagent solutions. The tentative procedure for the formation of sodium and potassium patterns is as follows: Zinc uranyl acetate solution serves as the reagent in the electrographic detection of sodium, a deposit of the sparingly soluble sodium zinc uranyl acetate forming on the plaster as the sodium ion migrates to the cathode. The pattern is rendered visible by examining the sensitized castin under ultraviolet light, which causes the sodium compound to fuoresce a bright green. It is not necessary to remove excess reagent, as the solids deposited on the plaster by the evaporation of the reagent solution are not fluoresCent.
I n general, when working with tissues of low alkali content it is necessary t o prolong the exposure for about 2 minutes in older to electrolyze a sufficient quantity of the ion being studied out of the section for the formation of a distinct image of the plaster medium. This results in a loss of definition owing to the diffusion of the mater-soluble reagents and the sparingly soluble precipitates. The analytical value of these patterns would be greatly enhanced by the discovery of fixed reagents for sodium and potassium following the principles formulated by Clarke and Hermance (1) for the use of such reagents in spot test analysis.
Summary h simple electrogiaphic method for the analysis of certain ionic constituents of plant and animal tissue is described, capable of rendering sharply defined patterns in terms of specific ions. The general technique has been improved through the use of sparingly soluble reagents distributed in ctructiireleqq media -uch a. gelatin and thin ea-tings of plaster
NOVEMBER 15, 1940
ANALYTICAL EDITIOK
of Paris. The improvements in the technique permit the observation of cellular detail in the electrographic pattern produced b y the chloride ion on a silver chromate gelatin emulsion.
Acknowledgment The writer wishes to express his gratitude to R *T' 'Ox for his kind cooperation in the design and construction of the electrographic apparatus and accessories described in this re-
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port. The writer is also indebted to R. F. Sigrelli for photographic assistance and to Alexander Gobus of Lucius Pitkin, Inc., for the photomicrographic reproductions.
Literature Cited (1)
Clarke, B. L., and Hermance, H. W., IND. ENQ.CHEM.,Anal. Ed.,
(2)
GIazunov, A., and K~ivohlavy,J., z. physik. Chem,, 161, 373
9, 292 (1937).
(1932). (3) Jirkovsky, R. J., Chem. ~ i s t y2, 5 , 2 5 4 (1931).
A Pyrex All-Glass Microelectrophoresis Cell DAVID R. BRIGGS Division of Agricultural Biochemistry, University of Minnesota, Minneapolis, Minn.
T
HE all-glass modification of the Sorthrup-Kunitz
microelectrophoresis cell (7) as described by Abramson (3) has proved very satisfactory in the measurement of migration velocities of particles suspended in aqueous media. It is not easy to construct, however, and its general use has been seriously curtailed on this account. I n certain cases, too, the danger of contaminating the liquid in the flat part of the cell by stopcock lubricant or electrode liquor may be of importance. A cell designed by Buzach and described by Bull (4) eliminates this latter difficulty but introduces others due to the fact that this cell is constructed by cementing the parts together. A recent discussion of the various modifications of microelectrophoresis cells can be found in a review article b y Abramson ( I ) . The microelectrophoresis cell and accompanying electrical hookup described here were primarily designed for use in measuring the migration velocities of particles suspended in organic liquids. The entire assembly may be used as well with aqueous systems. The simplicity of design and use and the relative ease of construction of the cell, together with the novel arrangement employed for determining the field strength during the measurement, seem to warrant publication of a description of this apparatus.
Design of Cell Figure 1 is a line drawing of the cell. The flat part, A , of rectangular cross section, is constructed from polished Pyrex glass slides, 25 X 75 X 1 mm. (1 X 3 inches X I mm.), obtained from the Corning Glass Company. Two such slides are ground
at the ends t o the shape shown in A' and are arranged in a parallel position with a spacer sheet of thickness equal to the depth desired in the finished cell (0.8 to 1.0 mm.) employed to hold the slides apart. (Sheet aluminum has been found usable for this purpose, but a slide of Pyrex glass similar to that used in forming the top and bottom of the cell is much better. A sheet of mica of the proper thickness should serve the purpose very well, since it would be resistant to heat and would not conduct heat away so rapidly as to cause strains to appear in the Pyrex slides.) The assembly may be supported in a vise with one long edge horizontal and extending well above the point of support. Careful adjustment of the distance between the slides (using a micrometer) at this point in the preparation will assure a minimum of deviation from parallelism of the top and bottom surfaces in the finished cell. The spacer sheet should extend about a third of the distance to the top edge of the assembly. A rod of Pyrex glass, approximately 2 mm. in diameter, is laid along the top edge of the assembly and melted on to the edge of the slides with an oxygen-gas flame (using a hand torch). The seal is extended along the ground ends of the slides. When the spacer sheet is removed the sealed edge supports the slides parallel to each other, By reversing the position of the slides in the sup ort, the other edge can be sealed in like manner. To the holes Eft at cither end of the flat cell is sealed a Pyrex tube of 8-mm. diameter. The slide which is to be the top of the flat cell is heated at the points of seal and blown to smooth connections with the tubes. The flat cell with its connecting tubes should be annealed carefully (by bringing it slowly to an incipient red heat in a muffle furnace and allowing it to cool slowly). The end parts of the complete cell, which should be prepared se arately and then sealed to the connecting tubes of the flat ceE, consist of the electrodes, B and B', the stopcocks, C, and, on one side, the funnel, D, through which the liquid will be introduced into the completed cell. Pyrex standard taper (So. 1) stopcocks, C, with I-mm. bore, present ample contact area to prevent leakage past these points when used either with or without lubricant. Each electrode is constructed from a Pyrex standard taper (12/30) ground-glass joint. The outer part, B , is sealed in, as illustrated, to form an integral part of one of the end parts of the cell. The inner part, B', may be sealed off at a point several millimeters below the joint and blown into the shape shown. The large ground contact area of the joint will prevent leakage at this point whether lubricant is used or not, and when the two parts of the joint are forced firmly together no other precaution need be taken to prevent the inner part from dropping out. I n order to use the cell with organic liquids i t is necessary to be able to remove the tvorking electrodes and change their contents easily, and to get the liquid into the flat part of the cell without danger of contamination from the electrodes or from stopcock lubricant. These requirements are met completely in the design as shown without introducing any trouble from leakage.
