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INDUSTRIAL .4ND ENGINEERING CHEMISTRY
It is interesting t o note t h a t 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 t o the blue-to-pink-to-colorless end-point change
VOL. 10, NO. 1
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 t h a t 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 t h e 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 and 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 t h a t individual r a t bones are photographed under a microscope
with lens equipment which gives approximately 5 X magnification. The method described i n 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 t o be much more economical than those previously described, both in materials and in the time required for the photographic processes. T h e results which may be obtained with this method are shown i n 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. T h e 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 a t 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 a t 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 x a s 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 a t 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
