ANALYTICAL EDITION
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with no resistance in the line was 1525’ C. and was reached after 3 hours. Higher temperatures could be obtained by the use of larger elements, but one is limited by the melting
Vol. 4, No. 2
point of the refractories. The atmosphere of the furnace chamber was found to be slightly oxidizing. RECEIVED
December 21, 1831.
Photographic Records of Vitamin D Line Tests HENRYST EVENS^ AND E. M. NELSON, Bureau of Chemistry and Soils, Washington, D. C. RAPID and economical method developed in this laboratory for obtaining photographs of vitamin D line tests has proved valuable as a means of permanently recording the results, as well as for illustrating published reports (1, 3 ) . The apparatus illustrated in Figure 1 consists of a small camera designed for direct attachment to the microscope stand. Focusing for a fixed magnification is accomplished through manipulation of the coarse adjustment of the microscope alone. Cameras of this type, differing considerably in certain optical and mechanical refinements, are made by a t least two manufacturers (Carl Zeiss, Inc., and E. Leitz, Inc.).
A
FIGURE 1. PHOTOQRAPHING APPARATUS
By use of an objective of 1 X2 t o 2 X magnification an image is obtained on a 4.5 by 6 cm. plate of approximately 5X magnification. This degree of enlargement permits the recording of one line test on each plate of sufficient magnification for precise interpretation. A plate of this size also has the advantage of economy both in cost of materials and in storage space. Incident illumination is provided by two Mazda, tubular, projection lamps (110 volts, 165 watts) mounted in porcelainlined, show-case reflectors. With this illumination, fully exposed negatives are obtained on panchromatic plates with exposures of from 1 to 3 seconds. The staining procedure followed in this laboratory conforms in general with the McCollum technic for the vitamin D line test (9). The animals are killed, and a radius is removed from each and cleaned a t once. The distal end of the bone is sectioned through the cqnter on a plane parallel with the flat surface where the distal ends of radius and ulna articulate. The two halves of the bode are at once immersed in water for 1 Working under fellowship of the National Cottonseed Products Association. The long workhg distance of this objective necessitated its attachment to the lower end of the draw tube of the microscope illustrated in Figure 1
about 15 minutes, or while an entire series of bones is being prepared for staining. They are then immersed for 1minute in 1.5 per cent silver nitrate solution, after which they are washed through two changes of distilled water to remove excess silver nitrate. The bones are then exposed in water to daylight or direct sunlight just long enough to develop a delicate straining of the calcified areas without darkening or yellowing the softer tissues. Descriptive records are then made, and the stained bones are stored in water in the dark until photographed. Undue delay in photographing often leads to difficulties, owing to degradation in contrasts through discoloration or unequaI swelling of the cut surface. For photographing, the bone is placed in the center of a 2inch (5.08-em.) watch glass and covered with a square cover slip. A sufficient quantity of boiled distilled water is run under the cover glass to just fill the space beneath it. Care must be exercised to avoid the inclusion of minute bubbles between the cover glass and the stained surface of the bone. A black background for the photograph is obtained by lowering the condenser and covering the under side of the stage with black paper from the wrappings of photographic plates. If the stained surface of the bone has become discolored through long standing or over-exposure to light, a better rendering of the calcified structure is ensured by interposing a suitable filter at some position between the object and the plate. The unmounted gelatin color filters are useful, as they may easily be trimmed to a suitable size and are not likely to upset the optical arrangement of the system. It has been found desirable in this laborator r filter, or its equivalent, on the up mount for all photographs of line te the contrast of discolored specimens without detracting from the quality of photographs of bones that are not discolored. If all exposures are made through the filter, estimation of proper exposure time is considerably simplified, because the filter introduces a constant factor in determining the length of the required exposure. As panchromatic plates are used exclusively, all manipulations in the dark room are carried out in total darkness. Rapid and uniform treatment of the plates is facilitated by carrying them through the various solutions and washing water in a developing rack carrying 10 or more plates. The plate rack shown in Figure 1, designed after the one used in the British “Dallon” developing tank, was made to carry 10 plates and fits into a container known as a 4 by 5 inch glass fixing box. This type of container is also used for the rinse or hardening bath and fixing solution. The shape of this container contributes to economy in the use of the developing solution and has the added advantage of a glass cover. Proper development is attained by adhering closely to a time and temperature schedule supplied by the plate manufacturer, the time being measured by an interval alarm timer. The developing solution used is that recommended by the plate manufacturer for high contrast. A quantity of this
April 15, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
solution sufficient for several months’ work, made up in twice the concentration used for developing, is stored in pint bottles. The photographs reproduce well either in half-tone or lantern slides, and they enlarge satisfactorily to several diameters. The procedure described can be successfully followed with little practice, and satisfactory records may be rapidly produced by technicians with little knowledge of the principles of photography. A modification may be made in the foregoing procedure which permits the bone section to be photographed by transmitted instead of reflected light, This modification involves a clearing procedure commonly used in the preparation of histological sections. After staining and washing the bone section as described before, it is dehydrated by successive
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15-minute immersions in 50 per cent, 95 per cent, and absolute alcohol. The section then transferred to xylene will “clear,” or become translucent, and may be photographed in xylene by means of a substage light source. Although this method may improve the rendering of structural details in some specimens, the results do not, in general, justify the additional time required when only laboratory records are desired. LITERATURE CITED (1) Manning, J. R., Nelson, E. M., and Tolle, C. D., U. S. Bur. Fisheries, Investigational Rept. 3, 1 (1931). (2) McCollum, E. V., Simmonds, N., Shipley, P. G., and Park, E. A,, Proc. SOC.Ezpptl. Bid. Med., 19, 123 (1921-1922). (3) Tolle, C. D., and Nelson, E. M., IND. ENG.CHEM., 23,1066 (1931). RECEIVED November 18, 1931.
An Apparatus for Electrodialysis E. J. KING,Department of Medical Research, University of Toronto, Toronto, Canada OST of the schemes for electrodialysis which have been described in the literature require special forms of apparatus which are often expensive and the parts difficult to replace. They usually consist of a cell of three chambers made of glass, earthenware, or rubber, and separated by membranes of various material such as pig bladder, collodion, parchment, parchment impregnated with gelatin, albumin, hemoglobin, and other proteins, and more recently membranes of cellophane. The most widely used apparatus for electrodialysis is probably that of Pauli (IO). It is a three-chambered glass cell fitted with electrodes of platinum or silver gauze, or graphite, and is obtainable on the market (Fritz Kohler, Leipzig), Other cells have been described by various workers in several papers appearing, for the most part, in the Biochemische and Kolloid Zeitschrifts (1, 8, 6, 9). Reviews of the work done with electrodialysis in colloidal and biological chemistry have been given by Dh6r6 (4), Rheinboldt (IS), Reiner ( I I ) , and Reitstotter (1.2). The theory of electrodialysis has been considered by these workers, by Ettisch (5) and Bradfield (S), and by others. Ettisch and his co-workers (5)have pointed out several dangers which may occur in the electrodialysis of blood serum. These include the possibility of temperature increases in the dialyzing liquid owing to the resistance of the cell to the passage of the electric current, and a tendency to precipitation of the proteins with the removal of the electrolytes and the changes in reaction which niay take place during electrodialysis. Figure 1 illustrates a simple scheme for electrodialysis, which iu very efficient and satisfactory for ridding colloidal and other solutions of electrolvtes in a minimum of time. The apparatus is simple and easjly constructed a t a small cost from material available in almost any biochemical laboratory. The semi-permeable membranes consist of 2 Schleicher and Schull parchment diffusion thimbles. The smaller inner thimble (1.6 by 10 cm.) contains an inlet and outlet tube for water and an electrode made of a strip of platinum sealed in a glass tube with a little mercury to make contact with the electric wire which is shoved down the tube. These three glass tubes pass through a rubber stopper which fits the smaller thimble after it has been soaked in water. The second and larger thimble (4by 10 cm.) contains the material to be dialyzed (about 50 cc.) and is fitted with a rubber stopper with a hole large enough for the smaller thimble to fit neatly into it. The diffusion thimbles, thus mounted, are clamped into
place so as to dip into a beaker which contains a pool of mercury in the bottom to act as the cathode. A glass tube runs down the inside of the beaker into the mercury and is held in place by a piece of adhesive tape stuck t o the side of the beaker. Through this tube, which must be sufficiently wide, an electric wire can be pushed down to make contact with the mercury. The inlet tube for water is connected by means of a rubber tube to the tap or distilled-water bottle. When
Mercury
J
FIGURE 1. DIAGRAM OF APPARATUS flowing, the water passes through the small thimble and drips into the beaker which, when full, siphons over to a constant level through the overflow tube, as illustrated. The electric current is passed by means of two leads dipping into their respective bits of mercury, the first making oonnection with the platinum anode and the second with the pool of mercury cathode. The amount of current passed is conveniently controlled by means of lamps interposed in series, parallel. Under the influence of the electric current, the ions of the electrolytes in the material to be dialyzed are carried to the electrode chambers, where they are washed away by the running water. The dangers referred to above are best avoided by passing a fairly weak current of electricity and by
