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It is interesting to note that the y intercept of the curve representing titrations against sodium thiosulfate is zero. The chief advantages of the proposed method are: The sodium thiosulfate solution remains stable after it has once reached equilibrium; the end point of the titration is sharp as contrasted to the blue-to-pink-to-colorless end-point change
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observed in the previous methods; and since standard sodium thiosulfate solution is normally required as a check on the standard iodine solution used in either of the other methods, the need for one of the usual standard solutions (the iodine solution) has been eliminated. R E C E ~ V E DSeptember
6, 1937,
A New Method for the Standardization of the Dye Used for the Determination of Cevitamic Acid (Vitamin C) ROBERT E. BUCK AND WALTER S. RITCHIE Massachusetts State College, Amherst, M a s s .
THE
new method which is presented is based on the fact that the dye, 2,6-dichlorophenolindophenol,will quantitatively oxidize iodide t,o iodine. The iodine liberated can then be determined by titration with standard sodium thiosulfate. This method is not only simpler in procedure than other methods, but also gives more accurate results. The results agree very closely with those obtained when pure cevitamic acid is used as the standard. ABSTRACTof paper presented before t h e Division of Biological Chemistry a t the 94th Meeting of the American Chemical Society, Rochester, N . P., September 6 t o 10, 1937. Copy of abstract received by -4. C. S . News Service July 30, 1937.
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Photographing Line Tests in Vitamin D Assays &I. WIGHT TAYLOR, DAIVIEL KLEIN,’ AND WALTER C. RUSSELL New Jersey Agricultural Experiment Station a n d Rutgers University, New Brunswick, N. J.
T
HE photographing of experimental results is by no means unusual in the field of vitamin D assays or in other lines
of work. Stevens and Selson (3) and Bacharach, Allchorne, Hazley, and Stevenson ( 1 ) hal-e reported the methods used in their laboratories for this purpose. The methods of these two groups of workers are fundamentally the same in that individual rat bones are photographed under a microscope
with lens equipment which gives approximately 5 X magnification. The method described in this paper permits the photographing of as many as 80 single radii on one 12.5 X 17.5 em. ( 5 X 7 inch) film or plate a t a 2 X magnification and is believed to be much more economical than those previously described, both in materials and in the time required for the photographic processes. The results which may be obtained with this method are shown in Figure 1, which is a portion of a typical photograph of the actual size used-that is, 2 X magnification.
Fixing and Staining Bones Although this paper is primarily concerned with the photographic technic, brief mention will be made of the prior treatment of the bones, since this treatment may influence slightly the final results. The method is, in general, very flexible and many modifications are possible. At the end of the assay period, the animal IS killed and the desired portions of the bones to be examined are removed and fixed in 95 per cent ethyl alcohol. It is preferable to allow the bones to remain in the alcohol for at least 24 hours for complete 1 Present address, Metabolic Laboratory, Philadelphia General Hospital, Philadelphia, Pa.
clearing, although this process may be hastened somewhat by splitting the bones before immersion in the alcohol. If an assay is not brought to completion in one day or if, for any otherreason, it is not convenient to make the photographs at the time of killing, the hones may be left in alcohol for an extended period of time. I t was found, for example, that the photograph of a group of left radii, made after the bones had been stored for one year in 95 per cent alcohol, showed calcification practically identical n-ith that s h o w by the picture of the corresponding right radii, made immediately after fixation. The use of lower concentrations of alcohol is not advised, since it xas found impossible to obtain satisfactory staining in the case of a few bones which had been stored in 70 per cent alcohol for less than 2 months. Ten per cent formalin is used by some workers as a fixative, but alcohol is preferred in this laboratory because it is not as unpleasant to handle, it yields a bone which is somexhat better for photographing, contrary to the observations of Bacharach et al., and the possibility of the leaching out of calcium salts by the more aqueous medium is avoided. Although no data have been obtained, the leaching effect might become noticeable if sufficient acidity developed from the formalin. It has been the practice in this laboratory to use only the distal end of the radius in making the line-test readings. However, since the radius and ulna are connected, the distal ends of the two bones are allowed to remain attached during the alcohol fixation. The most convenient procedure is to remove the distal ends of both sets of radii and ulnae, free the bones from the greater portion of adhering tissue, and tie both sets to a small paper tag bearing the rat number. The tying is done in such a way that one set of bones may very readily be removed, leaving the other set still labeled. This second set may be preserved for at least a year for possible future Kork. The bones are split and stained according to the regular procedure, except that care is taken to avoid overstaining. Usually from 50 to 80 bones are treated at one time. As each bone is split, it is immersed in water to remove most of the alcohol As soon as all the bones are split, the water is replaced by 1.5 per cent silver nitrate and the bones are stained by exposure to the
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ANALk-TICAL EDITIOS
ordinary diffused light of the laboratory. This lighting is recommended, since it gives a slow rate of staining and permits closer control of the depth of staining. Under the usual lighting conditions the bones darken sufficiently in 5 to 10 minutes. If the action is not rapid enough it may be hastened by means of artificial light. Actinic light, such as direct sunlight, a carbon arc, or one of the so-called sun lamps, will give very rapid staining, but has no particular advantage otherwise. As soon as the bones have stained so that a distinct, lightbrown image of the calcified areas can be seen, the silver nitrate is poured off and the bones are washed with three changes of distilled water. When treated in this manner the bones will darken only slowly on further exposure to light and, if protected from light by a layer of heavy paper, may be kept for 2 to 3 days prior to reading or photographing. If, however, the photographing is to be done immediately, it may be advisable to leave the bones in the silver nitrate until the staining becomes heavier. In some laboratories it is the practice to treat the stained bones with ordinary photographic hypo (sodium thiosulfate) in order to prevent further darkening, especially in the noncalcified areas, but in this laboratory the process has proved to be somewhat erratic. Since excellent results may he obtained without this step, its use is not recommended.
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the edge of one of these clumps, the clump often remains intact, all of i t appearing on one side and none on the other side of the bone. This may result in markedly different readings on the two sides of the same bone. Furthermore, it is apparent that, for the same reason, several sections through different parts of a single bone may give quite different pictures. Added to this fact, one has what may be called the normal variations of experimental animals. Hence, it would seem impossible to draw precise interpretations on the basis of a single line-test reading, and, although the use of such precision would not cause any additional error, i t would not introduce further accuracy. It is much more important that the treatment of the bones be as consistent as possible so that, if errors are present in the line readings of one group, the same errops in the same direction will be present in the readings of all the other groups.
Photographic Equipment For photographing, the authors use a Bausch & Lomb type H vertical photomicrographic camera equipped with a Bausch &- Lomb f 1:4.5 Nicrotessar lens of 48-mm. equivalent focus and a compound shutter. This equipment gives a minimum of two and a maximum of ten diameters enlargement. Most of the work in this laboratory is done a t the smallest degree of enlargement, since this allows a large number of bones to be photographed on one negative and at the same time yields a picture of the individual bones which shows adequate detail for the line-test readings. If larger pictures are desired it is comparatively easy to make photographic enlargements from the original negative.
FIGURE2. APPARATUSFOR HOLDIXG SPLITASD STAINED BONESFOR PHOTOGRAPHIKG For example, it is the practice in this laboratory, when the two sides of a bone show different amounts of calcium, always to photograph and read that half of the bone which gives the higher reading. Similarly, all the bones should be stained as equally as is possible and all bones should be photographed a t the same degree of enlargement.
-4rranging Bones for Photographing When the bones have become properly stained, the line tests may be read directly or the bones may be photographed and the readings made from the print. It is advisable to take a t least one set of readings on the bones to guard against the possibility of the disarrangement of their order. For photographing, the bone sections are arranged in r o w on a piece of blotting paper, backed by a glass plate, and are covered with another piece of glass, as shown partially in Figure 2. If large bones, such as the tibia, are to be photographed, additional layers of blotting paper may be advisable as a cushion to comFIGURE1 . ACTCALSIZEOF LISE-TESTPHOTOGR.~PHF pensate for uneven thicknesses of the bones. The sandwich thus formed is held together by rubber bands wound around each end. Unless there are marked differences in the thicknesses of the bones, no difficulty should be experienced in keeping all the Both Stevens and Selson ( 3 ) and Bacharach and co-workers bones in place. Using 2 X magnification on a 12.5 X 17.5 cm. (1) use an optical system which yields a 5 x magnification. (5 X 7 inch) film one may photograph an area of 6.25X 8.75 cm. It is stated by Stevens and Selson that this permits sufficient (2.5 X 3.5 inches). However, in order to provide a slight margin enlargement for precise interpretation. However, it would of safety so that all the bones will appear on the film, it is better to have the glass plate slightly smaller than the latter area. Lanseem that such preciqion is not qignificant when dealing with tern slide cover glasses cut in half to furnish an area 5 X 8.125 a single rat bone. This is particularly true when the line of cm. (2 X 3.25 inches) are excellent for this purpose. It is poscalcification is only partially formed, as then the calcification sible to arrange 8 rows of 10 radii each on this size of plate, and often occurs in irregular clumps. If such a bone is split near also to have space for writing the legends.
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FIGURE 3. APPARATUS FOR PHOTOGRAPHINO LINETESTS The color of blotting paper used as a backing for the bones is In this laboratory white is used; other workers prefer black. Possibly gray would be better than either, since it would contrast with both the white of the noncalcified area and the black of the calcified portion of the bones. No attempt is made to incorporate any legend with the actual bones. However, as shown in Figure 2, strips of black paper, 2 to 4 mm. wide, are placed on the blotting paper so that a strip occurs between each row of bones. The black strips appear as clear strips on the negative and furnish an ideal place for writing the rat number, line-test reading, and any other necessary information. This material is written on the reverse or shiny side of the film with India ink, using a fairly fine pen point, and appears as white letters on a black background in the final picture. In addition, a portion of a thin metric ruler is placed under the cover glass at one end of the assembly so that the degree of enlargement may always be verified. All the bones of a single test are arranged on one line, so that it is very easy to cut the print into strips. After the bones have been arranged and the cover glass has been fastened in place, the whole assembly is slowly immersed edgewise in a Petri dish of distilled water, care being taken that no air bubbles are trapped between the glass plates. Sufficient water is then added to cover the upper glass surface completely, in order to eliminate the glass-air surfaces which might cause undesirable reflections. The use of a polarizing screen (sold under the trade name Pola Screen) to lessen reflections in large areas or from tiny spots has been recommended (2). However, little difficulty should be experienced, if the direct rays from the lights strike a t an angle which does not reflect into the lens. The screen might prove to be of value if extremely fine detail were desired in the picture. a matter of individual choice.
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The usual method of focusing is by means of a focusing mount which carries the lens, but in most of the work in this laboratory it has been considered desirable to make all the photographs at a fixed degree of enlargement. Hence, the position of the camera and lens is kept exactly the same and focusing is accomplished by moving the object to be photographed. For this purpose a table, adjustable in height, made by fastening a light board to one of the cup holders of a discarded colorimeter, has been found convenient. It is, of course, important that the improvised support be perpendicular to the axis of the camera and lens in order to avoid distortion of the image. After focusing, the lens diaphragm is shut down to f 11, the film holder inserted, and the picture is taken. With most lenses there is no point to stopping down the lens further, since the resolving power of lenses tends to decrease with small diaphragm openings. The correct exposure is important, but since it will vary with the type of film, the intensity of the light source including the general illumination of the room, the degree of enlargement, and the object being photographed, no definite time can be given. However, as a guide, with two 60-watt lights in small reflectors about 15 cm. (6 inches) from the object, using commercial panchromatic cut film, and enlarging 2 X, an exposure of 10 seconds at stop f 11 has been found adequate. Any other film may be used, but the low-speed, high-contrast film such as process, process panchromatic, commercial, or commercial ortho will be found best for most subjects. If there is a tendency for the uncalcified portions of the bone to acquire a reddish stain, the panchromatic films should be used. In most cases the orthochromatic films are preferable, since development may be carried out in red light. Development of the film and of the subsequent prints should be carried out according to the manufacturer’s directions, although it has been found convenient in this laboratory to use the same developer (Eastman Kodak Formula D 72) for both film and paper.
Summary The technic of staining and photographing rat bones for records of vitamin D assays is described. The apparatus and technic employed allow the photographing of as many as 80 single rat radii on a 12.5 X 17.5 cm. (5 X 7 inch) film a t 2 X magnification.
Literature Cited (1) Bacharach, A. L., Allchorne, E., Hazley, V., and Stevenson, S. G., IXD.ENG.CHEM.,Anal. Ed.,5, 12 (1933). (2) Stevens, H., private communication. (3) Stevens, H., and Nelson, E. M., IND.ENO.CHEM.,Bnal. Ed., 4, 200 (1932). RECEIYED August 5, 1937. Journal series paper of the New Jeisey AgriExperiment Station, Department of Agricultural Biochemistry.
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Lighting
The light source should be fairly brilliant for ease in focusing and well diffused, since the black and white of the bone surface is the only thing of interest. The practice in this laboratory is to use two 60-watt bulbs in small reflectors, which are supported b y a ring stand and clamps so that a light is on opposite sides of, and a t the same height as, the camera lens. This arrangement gives some shadows, especially between the bones, but they are not confusing. The complete equipment, including the lights, is shown in Figure 3. Additional lights might be used to form a complete ring which would yield a stronger light with less shadow effect, but the advantages do not seem commensurate with the work required. Photographic Technic With the bones in place and the lights adjusted, the image is focused on the ground glass with the lens diaphragm wide open.
ULTRAFILTRATIOS PROCESS, LILLY RESEARCH LABORATORIES
