View source for Book - A text-book of histology arranged upon an embryological basis (1913) 2-2

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<center><big>'''Part II. Microscopical Technique'''</big></center>

=II. The Examination of Microscopical Specimens=

==The Microscope==

It is unfortunate that the price of a microscope is prohibitive to many medical students, and that some who might purchase instruments at the beginning of their work wait until later. The cost is now so reduced that an increasing proportion of students can enjoy the advantage of having a microscope of their own.

Microscopes of a certain grade are required, and if they cannot be afforded, no instrument should be bought. The necessary equipment, as shown in the figure, is a stand with fine and coarse adjustments ("micrometer screw" and "rack and pinion"), and a large square stage. The more expensive round and mechanical stages are not necessary, and since mechanical stages are detachable, they may be obtained later if desired. There should be an Abbe condenser (with iris diaphragm), a triple revolver, a high and a low eyepiece or ocular, and the following objectives: a i6-mm. (f-inch) and a 4-mm. (|- or f-inch) which must be parfocal; together with a 2-mm. (yV-inch) oil immersion, for cytological and bacteriological work; and a 48-mm. (2-inch), which is a very low power, for embryological work. The figures indicate the distance of the section from the objective when the specimen is in focus; the higher the power, the nearer the objective is brought to the object. The 2-mm. oil immersion is an expensive objective, and its purchase may be postponed . The 2-inch is a cheap objective which is very useful in obtaining a view of an entire section, and for embryological reconstructions it is essential. It may be noted that microscopes are now being finished more extensively in black enamel than in lacquered brass; the former is not damaged by alcohol and is more desirable. Improvements have also been made in the post and fine adjustment, so that the form shown in the figure, although good, is not the best.

Satisfactory microscopes of American manufacture are now made but all agree that the Zeiss microscopes (German) are the best (and most expensive). If the microscope is purchased by a student unfamiliar with its use, it is well to have the lenses' iacamined by a disinterested microscopist.

For a description of the nature and use of the microscope, the student is referred to the nth edition of "The Microscope," by Professor S. H. Gage (Comstock Pub. Co., Ithaca, N. Y.).


For the sake of emphasis it may be said that the microscopist works with his right hand upon the fine adjustment and his left hand upon the slide. As the latter is moved about, bringing different fields into view, the focussing is done with the adjustment and not with the eyes. It is impossible to study even a single field without constantly changing the focus, and the continuous use of the fine adjustment distinguishes an experienced microscopist from a beginner. Both eyes should be open (as will be natural after becoming accustomed to the instrument). Often one acquires the habit of using only the right or the left eve for microscopic work, but it is better to learn to use both.







FIG. 493.




Always examine a specimen first with a low power objective and then with a high power. In focussing the microscope, have the objective drawn away from the slide and focus down. This should be done cautiously, with a portion of the specimen actually beneath the lens; if there is only cover glass and damar there, the objective will probably be driven down upon the slide. Unless one is sure that stained tissue is in the field, the slide should be moved back and forth as the objective is being lowered.

In working with the Abbe condenser, the flat surface of the mirror should be uppermost, provided that it is used in daylight and the rays falling upon it are therefore parallel; but for the divergent rays of an artificial light near at hand, the concave mirror may be used, and the light may advantageously be made to pass through a blue glass, which lessens the yellow glare.

The objectives must never be scratched. Lens paper or fine linen should be used to wipe them. If they are soiled with damar they should be wiped with a cloth moistened with xylol, but since the lenses are mounted in balsam, xylol must be applied to them cautiously. A microscope of the kind shown in the figure should never be lifted by any part above the stage, lest the fine adjustment be damaged; the pillar should be grasped below the stage.

==Reconstructions==

There is an important arrangement of mirrors (Abbe's camera lucida) for drawing the outlines of sections. It is attached to the microscope above the eye-piece, and on looking into it one can see the image of the section beneath the objective apparently spread upon the drawing paper beside the microscope. Thus the pencil point can be seen as it is made to trace the outline on the paper. With a little practice the same result may be obtained more or less perfectly without the camera, by looking into the microscope with one eye and at the same time upon the paper with the other. This possibility was noted by the early microscopists, and it is a useful accomplishment. More satisfactory than the camera lucida is the projection apparatus of Edinger, arranged with an arc light, whereby the image of the section is projected through an inverted microscope upon the drawing paper beneath. With the camera, or projection apparatus, a succession of serial sections may be drawn with the uniform magnification essential for reconstructions. The magnification is determined by substituting a stage micrometer for the slide of sections. The micrometer is a slide upon which i mm., with subdivisions into twentieths or hundredths, has been marked off by scratches in the glass; the subdivisions may be drawn with the camera, under the same conditions as the sections, and the enlargement of the subdivisions may then be measured.

From the camera-drawings of serial sections, wax reconstructions of various embryonic organs or small structures in the adult can be built up. If the sections are 10 /* thick and alternate sections have been drawn, magnified 50 diameters, then, on the scale of the drawings, these alternate sections are i mm. apart. Wax plates i mm. thick are therefore to be made, either by rolling beeswax, or by spreading a weighed amount of melted wax in a pan of hot water. It floats and spreads in an even layer, solidifying as the water cools. The outlines of the drawings are then indented upon the wax plates, and the desired portions are cut out and piled up to make the model. In this way reconstructions like those of the ear (p. 466) may be made. This method was first employed by Born. Further details of the process should be learned from demonstrations in the laboratory.

Graphic reconstructions (first used by His) are generally side views of structures, made from measurements of their transverse sections. Fig. 176, p. 185, is from such a reconstruction. A camera drawing of the side of an embryo (or other structure) is made before it is sectioned. The outline of this drawing is enlarged, and parallel lines, equally spaced, are ruled across it, corresponding in number and direction with the sections into which it was cut. Often only every other section, or every fourth section, is used for the reconstruction, and the number of lines to be ruled across the drawing is correspondingly reduced. Camera drawings of a lateral half of every section to be used in the reconstruction are then made, and across each drawing two lines are ruled. The first follows the median plane of the body; and the second is at right angles with it, being drawn so as to touch the dorsal or ventral surface of some structure to be included in the reconstruction. Provided that the camera drawings and side view have been enlarged to the same extent, the perpendicular distance from the middle of the back to the junction of the two lines is marked off in the side view, on the line corresponding with the section in question. The perpendicular distances from the second line to the dorsal and to the ventral surfaces of all structures to be reconstructed, are also marked off upon the line on the side view. The same is done in the following section, and the points belonging with a given structure are connected from section to section. Thus the outlines of the organs are projected upon the median plane; two dimensions are accurately shown but the third is lost.

Often it is undesirable to attempt to make the magnification of the sections and of the side view identical; the measurements may be enlarged or reduced as they are transferred for plotting, by means of the draughtsman's proportional dividers, an indispensable instrument for this method of reconstruction. The corrections for unequal shrinkage of the sections in paraffin, and other details, can best be explained in the laboratory with the drawings at hand.

In addition to making side views, this method may be used in reconstructing ventral or dorsal views, by plotting outward from the median line.

==Drawings==

Since anatomy, both gross and microscopic, is a study of forms and relations, that is of things seen, it finds natural expression in drawing; and the volumes of wood-cuts, copper-plates, and lithographs, together with the cheaper process-drawings and half-tones of the present day, form almost as important a part of anatomical literature as the accompanying text. Often there may be shown in a figure at a glance what pages of writing fail to make clear; and it is significant that the great books of Vesalius, which marked a new era in anatomy, were illustrated by Jean de Calcar, a pupil of Titian. Burggraeve believes that Vesalius doubtless supplied preliminary sketches and adds "Almost all the great anatomists were no less excellent draughtsmen Scarpa and Cuvier furnish us remarkable examples and one can hardly imagine an anatomist who is not deeply sensitive to the beauty and harmony of contours and forms." Selenka (1842-1902) drew the ape embryos, which he collected and described, with consummate skill, and "always impressed upon his students the great value of a ready pencil." Robert Hooke (1635-1703) was far less successful with his drawings. In the preface to his fully illustrated microscopical observations, he makes the following explanation of the defects of his plates, and in conclusion sets an example which all students should follow. He says:
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"What each of the delineated Subjects are, the descriptions annext to each will inform, of which I shall here, only once for all, add, that in divers of them the Gravers have pretty well followed my directions and draughts: and that in making of them, I endeavoured (as far as I was able) first to discover the true appearance, and next to make a plain representation of it."
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To discover the true appearance of each section and to make a plain representation of it, is by far the best method for beginning the study of histology, and conscientious attempts to represent what is seen invariably lead to deeper and more valuable observations. Thus drawings are unhesitatingly required of all students, and every effort should be made to acquire some skill in this direction. The problem of the microscopist, who has but little to do with the third dimension, is relatively simple. A few suggestions may be given.







FIG. 494. DIAGRAMS SHOWING THE WAY IN WHICH THE SHADE VALUES OF THE PRIMARY SECONDARY AND TERTIARY COLORS MAY BE REPRESENTED IN TERMS OF BLACK AND WHITE (Lee, in Hardesty's "Laboratory Guide;" Blakiston, 1908.)

Generally sections are stained in different colors, and the question at once arises how to represent these with the pencil. The accompanying figures indicate the way in which this is done, the primary colors being shown in the inner ring, and their combinations in the outer rings. Red being a brighter color than blue is to be made lighter. Orange, a combination of the two brightest of the primary colors, should be lighter than purple a combination of the darkest and lighter than pure red since it has the brighter yellow mixed with it. Thus the various colors may be suggested in black and white, and the contrast between blue nuclei and red protoplasm can be carefully preserved in the drawing. This is facilitated by the use of pencils of varying degrees of hardness "H" and "3 H" for dark structures, and "6 H" for pale areas. Soft pencils, which rub, should not be used.


Before beginning a drawing, the specimen should be carefully looked over, to find the place most worthy of such attention. The time which the drawing is to take must be considered, and a small area may be found which combines features elsewhere scattered about the specimen. The entire field is rarely, if ever, to be drawn; and the figures should not be encumbered with surrounding circles.

The magnification of the drawing is next to be decided upon. The form of a gastric gland and the structure of its cells, for example, cannot profitably be included in a single drawing. General features, such as the forms of glands, should be represented in "low power" sketches. "Low power" as here used does not necessarily refer to the lenses employed, but means that the drawing is on such a scale that the nuclei appear merely as spots, round or elongated as the case may be. Often, however, such a drawing shows features which can be clearly observed only with high power lenses. "High-power drawings" are those which present details of nuclear and protoplasmic structure.

Usually in studying an organ, it is desirable to make a general lowpower sketch showing the arrangement of its lobules or layers, and to supplement this by high-power drawings of the most significant cells or tissues. In these, which are the final test of a student's keenness of observation, no details of cellular structure are too minute for careful representation, and "the difficulty of observing them proves not the merit of overlooking them."

Having selected a field and decided upon the magnification, the outlines of the parts should be sketched lightly, with a soft pencil, and corrected until accurate. As finally made, they should be definite clean lines, not pieced out, representing the boundaries of layers, nuclear membranes, cell walls when present, cuticular surfaces, and the like. Having completed the outline, shading should be undertaken, to differentiate substance from empty space, and to indicate the nature of the substance. In highpower drawings protoplasmic texture must be faithfully reproduced homogeneous, finely granular or coarsely granular; if the granules are not distinct enough to be counted, they should not be readily countable in the drawing. If definite walls are absent from the specimen, they should not be drawn, but the shaded areas of the finished drawing should end abruptly without a bounding line.

Drawings consist, therefore, of two parts outline, and shaded texture or finish. Ruskin observes that the real refinement of the outline depends on its truly following the contours, and in regard to finish be offers suggestions which may be applied to the drawings of the wall of the medullary tube here reproduced. He states that if we are to "finish" farther, we must know more or see more about the object. These sketches are not finished in any sense but this, that the paper has been covered with lines. A piece of work is more finished than others, not because it is more delicate or more skillful, but simply because it tells more truth. " That which conveys most information, with least inaccuracy, is always the highest finish."






FIG. 495. THE WALL OF THE MEDULLARY TUBE, AS DRAWN BY Six STUDENTS.



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