ANALYTICAL EDITION
12
feel the material push ahead of the wooden roll, and the proper spread can be accomplished without separating the plates. It is advisable to separate them partly to check the spread until one becomes accustomed to the method. The rubber roll should be longer than the plates are wide, and the wooden roll 1 to 2 inches (2.5 to 5 cm.) in length. The wooden roll must be used only after the rubber roll or the plates will begin to dish. Results indicate that if constant weight is not attained in 2 to 4 hours, the rolling was not sufficient. When no further spreading is possible, the plates are pulled apart and placed in the drying oven. When it is necessary to weigh, they must be placed together as quickly as possible
Vol. 5, No. 1
with the sample side in, and slipped into the frame and cooled in a desiccator. Cooling as well as heating is very quick. The final most satisfactory equipment was obtained from Eimer & Amend. LITERATURE CITED (1) Balch, R.
T.,J . Assoc. Oflcial Agr. Chem., 15, 176 (1932). (2) Rice, IND. ENO. C H E X . , 21, 31 (1929). RECEIVEDAugust 2, 1932. Presented before the Division of Sugar Chemistry a t the 82nd Meeting of the American Chemical Society, Buffalo, N. Y., August 31 t o September 4, 1931.
Photographic Recording of Line Tests for Vitamin D A. L. BACHARACH, E. ALLCHORNE, V. HAZLEY, AND S. G. STEVENSON Glaxo Research Laboratory, 56 Osnaburgh St., London, N. W. 1, England
S
TEVENS and Nelson (1) have recently reported a
method developed in their laboratory for keeping permanent records of vitamin D line tests. For the last four years a photographic method, developed independently of theirs and depending on the same general principles, but differing in a number of details, has been used in this laboratory. The possibility that some workers may find these alternative methods better adapted to their conditions than I
IO ‘m
I
per cent formaldehyde solution until it is wanted for further treatment. Bones cleaned of blood in this way with formalin give somewhat clearer lines than those cleaned with alcohol. Owing to the frequent shortage of bright sunlight in London, bones are usually stained a t about 12 inches (30.5 cm.) from a Hanovia 220-volt mercury arc for a period of about 60 seconds. After being washed, the bones are %xed” for a few moments in 25 per cent sodium thiosulfate solution. This seems to make the staining more permanent, and is a convenience if photography is to be postponed for some days. The process must, however, be watched, as too prolonged a treatment tends to “unstain” the bones. Figures 4 and 5 are of bones that had been stained and “fixed” 4 weeks before. In other respects this treatment of the bones is essentially the same as that used by various other workers.
FIGURE1. PLANOF MICROSCOPE
STAND
H ! hole, and G, groove, for receiving feet of triangular platform; outline of triangular platform and leveling screw8 in place shown by dotted lines.
are those of Stevens and Nelson led to the publishing of this report. As a matter of convenience, the stages of procedure will be described in chronological order. PREPARATION OB BONE
As it is sometimes necessary to keep the bones for a few days before they are stained, as, for example, when several animals are being taken off one test on different days, the cleaned, unsplit bone is simply dropped into neutralized 10
FIGURE2. PHOTOMICROGRAPHIC SET F foousing eye-piece, C : condenser for “Pointolite” lamp N, holder for color screen D ,glass dish for holding specimen
P, paper record of animal number
(not in place) T, trlangular p l a t f o r m with leveling screws
S, color screen
January 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY THE OPTICALSYSTEM
An ordinary Zeiss microscope stand, with coarse and fine adjustment, a Watson "Halos" 3-inch (7.6-cm.) objective, and a LeitZ Periplan 0. K*six-times eyepiece are used. Instead of fitting the objective to the lower end of the drawtube, as do Stevens and Nelson, it seems best to remove the
13
By the use of the Leitz camera an image is obtained in a circGlar field, the diameter of which justcovers the shorter dimension of a 4.5 by 6 cm. film, The photographs a magnification of 5 to 6, and are therefore larger than those obtained by Stevens and Nelson, although the films used are the samesi~eastheirplates.
THEILLUMINATION 3 .O
0
2.5
0 2.0
2. 1.5
3. 4.
x
..4-
v)
6.
e. 1.0
IO
6
20
30
OS
40
60
--
IM 00
0 4SO
5 00
550
6 00
e50
700
750
Wave le n gt h
A single Ediswan “Pointolite” 100-250-volt, 100-c. p. lamp is used facing the observer, in a specially constructed stand, with a small condensing lens; the whole arrangement is shown in Figure 2. The lamp throws a shadow of the specimen between it and the observer, but this is not a disadvantage, as it gives a certain realistic stereoscopic effect to the photograph (see Figures 4 and 5).2
THEFILM The use of film packs, apart from any question of convenience in handling, is an advance in safety and economy of storage. Films take up much less room than plates, and are unbreakable. The Imperial Panchromatic film pack has been found ideal.
Courtesy Meesre. IZford, Ltd., England
THE FILTER
FIGURE 3. “ILFORD” MICROFILTER No. 5 TRANSMISSION CURVE
whole of the microscope stage and place the specimen to be photographed either on the bench, or on a little triangular platform fitted with an adjustable leveling screw a t each corner. One of these screws fits into a small hole on one side of the microscope foot, the other into a groove on the other; the third foot is apparently free, but actually has no degrees of freedom, and the platform is therefore quite firm during focusing and exposure (see Figure 1). The camera attachment by Leitz has a side-focusing ocular, but unfortunately this can be used only as a “spy-hole,” to verify the position of the specimen and the illuminated circle in the microscope field, and not for actual focusing, which must be done on the ground-glass screen of the camera. Owing to certain optical effects, the image given by the ocular appears sharp over a much greater range of the microscope adjustments than does the image of the screen. The manufacturers have informed the authors that this trouble is inherent in the use of the camera for low magnifications. The accommodation of the eye adjusts automatically for minor errors in focusing by the ocular, whereas these errors can be detected on the ground-glass screen. The ocular, though suited to medium and high-power work, cannot be used to maximum advantage for low magnifications without alteration of the optical system If, however, this defect could be rectified, a very great saving of time could be made, owing to the use of film packs, as described below. The manufacturers suggest that the only way of overcoming the difficulty is to fit a small lateral ground-glass screen in a funnel-shaped tube with focusing magnifier. The plane of this screen would be a t right angles to that of the camera ground-glass screen, but the length of the optical axis would be identical. By focusing on the image the lateral ground-glass screen, perfect focus would be obtained with even the lowest power 0bjectives.l 1 Since this paper was written, the authors have received from Messrs. E. Leitz a camera attachment with the ocular redesigned on the suggested lines. It behaves exactly as the manufacturers foretold, with the further advantage t h a t i t permits the image t o be focused without removal of the orange color dlter, thus making a reduction in the number of necessary movements by the operator A routine worker, using the new attachment
Originally a green color filter, recommended by the Imperial Dry Plate C o m p a n y , was used; more recently the Ilford micro 5 orange filter has been found to give even better contrast, with no sacrifice of detail, and allows a c o n s i d e r a b l y s h o r t e n e d exposure. The K3 Wratten filter mentioned b y 8 t eve n s and Nelson is deep yellow and probably similar in optical properties t o the micro 5. Using the develop er mentioned below, the correct exposure was found to be as follows: Ex-
FILTER POSURE K3 Micro 5
DEVELOPMENT
Sec.
Min.
15
16 15
2
FIGURE 4. PRINTFROM WHOLE NEGATIVE Showing confines of illuminated area and typewritten animal serial number corresponding with specimen G332.
The print reproduced in Figure 4 was made with the short exposure and the orange filter, those in Figure 5 with the longer exposure and green filter. All exposures have been made with the center of the and film packs of 12, can photograph 12 lines in 20 t o 25 minutes. The camera-attachment is, the authors are informed, obtainable from any of the branch houses of the manufacturers. * Owing to the different relative positions of the axes of camera and ocular in the new apparatus, i t has been found more convenient t o work with the lamp to the right of the operator, the microscope and camera immediately in front of him, and the ocdar pointing towards him. The image of the object so obtained is seen sideways by the observer, instead of upside down, as before.
ANALYTICAL EDITION
14
“Pointolite” bulb about 17 cm. from the specimen. Modern films have very great latitude, and the precise exposures may vary considerably without seriously affecting the quality of the negative, but the same exposure and development should be given to all films, if it is desired also to make prints with a constant exposure. For development, which takes place in a Kodak tank after removal of the backing paper from each film, a 10 per cent Rodinal solution is used for 15 minutes a t 19” to 20” C.; the films are fixed in any good acid fixing bath, and washed and dried in the usual way.
G231
G228
G229
FIGURE 5 . TRIMMED FINISHED PRINTS G232, severe rickets G228, marked healing G229, advanoed healing
Contrary to Stevens and Nelson, the authors have a preference for a white background on the print. Each specimen is therefore placed in a flat-bottomed thin glass dish, which in turn rests on a piece of white paper, and on this is typed the serial number of the animal whose bone is being photographed. This number thus appears photographed on the negative-a feature that, for recording purposes, is of outstanding importance. If a black background is preferred, a strip of white paper with the number typed on can, of course, be used. The typing is placed in such a position relative to the specimen that it can be trimmed off the finished print without trimming off any of the essential part of the bone photograph (see Figures 4 and 5). The specimen is just covered with water, and kept submerged by gently pressing the broken end of the shaft (which does not appear in the photograph) into a small piece of plasticine; this may, if desired, be fixed onto the bottom of the dry dish. If a series of photographs is being taken, it is important to change the water in the dish quite frequently; otherwise, owing to the heat from the lamp, steaming may affect the sharpness of the image. The choice of film packs rather than plates was determined by another consideration. The authors had hoped to focus each specimen solely by means of the ocular. I n that case, after each exposure the next film in the pack would simply have been brought into position without removing the film pack adapter from the camera, and the next specimen placed under the microscope and focused; as it is, the film pack adapter has to be removed and each specimen focused afresh on the ground-glass screen. If the trouble with the focusing ocular could be overcome, a t least a 50 per cent saving of time could be effected in photographing a series of six or more lines.’ Development in total darkness is secured by means of the developing tank, but the usual green safe-light can be discreetly employed while the pack is being unloaded and the films transferred to the tank. PRINTING
If the negatives are of the right quality, “vigorous” or L‘contrasty” paper should not be necessary for the prints, which can be made on any good quality medium or normal
Vol. 5, No. 1
gaslight3 paper, developed with amidol and fixed, glazed on glass or on ferrotype, and dried in the ordinary way. Glossy paper, whatever its esthetic disadvantages, is used in deference to the wishes of block-makers, who prefer it; the photographs lend themselves admirably to halftone reproductions (Figure 5. See also Winton and Bayliss, 2). The finished prints from one test can either be mounted on a card to bring together the results of the test, or stored in a small envelope, as can the negatives, even when as many as twelve refer to a single test. It is convenient to keep a card of mounted prints along with the line-test record card (Figure 6). and also a spare set of prints for each test. betails of the test can be entered on the envelope of films as well as on the record card. Certain investigators state that they prefer the method of pen-drawing or the use of the camera lucida to actual photographic recording, even, in one case, after a considerable trial of the latter. Their preference is probably based on their experience that uncalcified portions of the bone which have darkened for irrelevant reasons-incomplete removal of blood, over-staining of the whole bone, etc.-are recorded by the camera in the same way as the calcified portions normally stained with silver, whereas the pencil or the camera lucida drawing can record the allowance made by the observer for those defects of staining technic which are never wholly absent. The authors agree with Stevens and Nelson that correct photography should distinguish between the true black-stained calcified tissue and other darkened portions of the bone. I n
D
-
on Bnral Dcrl :
-
-
T on Ted : K Killrd,
FIGURE 6 . LINE TEST RECORDCARD
our experience it is only the rare, extreme case in which the photographic record does not allow a t least as accurate a diagnosis as that made by direct observation, provided the photograph has been taken with a technic adapted to secure the proper color corrections, by the use of panchromatic films or plates and a suitable filter. LITERATURE CITED (1) Stevens, H., a n d Nelson, E. M., IND. ENQ.CHEEX., Anal. Ed., 4, 200 (1932). (2) W i n t o n , F. R., a n d Bayliss, L. E., “ H u m a n Physiology,” Figure 97, p. 233, Blakiston, 1931. RECEIVE~D June 21, 1932. 8 The term “gaslight paper” is used in Great Britain for slow developing paper, .of such speed that a normal negative requires from 5 t o 15 seconds’ exposure a t 12 inches from a 50- to 100-candle-power lamp, and 1s fully developed in 30 to 60 seconds.
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