Illumination is one of the most important points in practical microscopy. With powers of not less than 1/2-in. focus, objects may generally be examined by light thrown upon them from above by a bulls'-eye condenser, or by good daylight. In this case they need not be transparent; and the plan is often convenient for a mere surface examination. In examining bodies illuminated in this way, prominences often appear as hollows and vice versâ, by a sort of optical illusion, which, once established, is very difficult to overcome. By remembering the direction of the light, and that this appears reversed in the microscope, it is easy to decide the truth.
For all finer work and higher powers, and most generally with the low powers also, it is necessary to render the object transparent, and to examine it by light transmitted from the mirror below the stage.
Good daylight is least trying to the eyes. Where artificial light must be used, that of a small paraffin lamp is best; and a blue chimney, or blue glass interposed between the stage and mirror, or lamp and microscope, spares the sight, and makes it easier to distinguish colours. The light should be sufficient, but not too dazzling. Work should never be prolonged after the least strain is felt, nor should the microscope be used for some little time after a meal. It is well to accustom oneself to keep both the eyes open while observing.
If it be required to see how far the cellular structures of the hide, such as hair-sheaths and fat-glands, are affected or destroyed in any stage of liming or bating, the following ready method may be employed. If a strip of hide be cut 2/3 through from the grain side, as shown at a in [Fig. 7], and the flap be turned down, and held between the finger and thumb, the fibrous tissue will be put on the stretch, and will then allow a moderately thin shaving (including the grain and parts immediately below it) to be cut by a sharp razor. The hide should be held in the positions shown, and a steady drawing cut be made from flesh to grain, the razor being steadied on the tip of the forefinger, and its hollow surface flooded with water. If the thin section be now placed on a glass slide, moistened with a drop of water, and examined on the microscope under a strong light from above, with a 1-in. objective, the fat-glands will be seen as yellow masses, embedded in the white fibrous tissue. If a drop of a mixture of equal vols. of strong acetic acid, glycerin, and water be used to moisten the section, the fibrous tissue will become quite transparent, and whatever remains of the cellular tissue will be easily visible, and may even be studied under tolerably high powers if covered with a thin glass, and lighted by the mirror from below. (The cover-glass must be carefully cleaned by rubbing with a linen handkerchief, and placed in position with a pair of tweezers, one side being supported by a needle, which is gradually withdrawn, so as to avoid air-bubbles.) Care must be taken that this mixture does not touch the brass-work of the microscope; even the vapour is apt to tarnish, so that the preparation must not remain longer than necessary on the microscope. The same method is applicable for ascertaining the completeness of the tannage of leather, and to decide whether the hide fibre is really tanned, or only dyed. Actually tanned leather is unaffected by the acetic acid, but raw or only stained hide swells and becomes transparent.
Fig. 7.
To prepare the very thin sections necessary for detailed study of the hide, more complicated methods are required. Small slips of hide, not exceeding 1/4 in. wide, and cut exactly across the lie of the hair, are placed first in weak alcohol (equal parts methylated spirit and water), and, after a few hours, are removed into strong methylated spirit. It is then kept for some days in absolute alcohol, which must be repeatedly changed, until the hide is hard enough to give fine shavings, and may be cut either when held as above described, between cork or pith, or when embedded in paraffin wax. This is accomplished by placing the piece of hide in a little paper-box and covering it with melted paraffin (candle), which is just beginning to stiffen. The piece of hide may be fixed in position with a needle, which must of course be withdrawn before cutting. When hard, the paraffin is shaved away till the object is exposed, when it may be cut. The razor must be wet with alcohol, and the section be made exactly in the plane of the hair-roots, which may be seen with a hand-lens. (The use of a microtome for hide-sections is rarely successful, as it is almost impossible to fix the fragment of hide so that it is cut exactly with the hairs.) The slices may now be stained by placing them in a watch-glass with water and a few drops of the logwood or picrocarmine staining-mixtures sold by opticians, and afterwards either examined in glycerin, or, after soaking some hours in absolute alcohol, may be transferred to clove-oil, and afterwards to a slide, and covered with a drop of dammar varnish or Canada balsam dissolved in chloroform. The sections moistened with glycerin may also be mounted in Farrant's solution or glycerin jelly, under a cover-glass for permanent preservation. If picrocarmine be used, the connective-tissue fibres (gelatinous fibres) and the nuclei of the cells will be coloured red, and the cells themselves of both epidermis and glands, together with the muscles and elastic fibres, will be yellow.
Franz Kathreiner, who has made very elaborate researches on skin, and the changes which take place in it during the processes of tanning, employs a mixture of osmic and chromic acids for hardening, and at the same time staining the tissue. This mixture was first used by a German histologist with whose name I am not acquainted, in a research on the internal organs of hearing, and was applied by Kathreiner in 1879 to the investigation of skin, and communicated by him to the writer in the autumn of that year. His method is briefly as follows. The pieces of hide to be examined must, if salted, be well washed, or if dry, be thoroughly softened. For the study of hide in its unaltered and natural condition, it is essential that it be quite fresh, and taken from the animal as soon as possible after death. In any case the Panniculus adiposus or fatty layer is, as far as possible, removed with scissors, the hair cut short, and the skin cut up into little pieces of 3-4 millimetres wide by 10-12 millimetres long (about 1/8 in. by 1/2 in.); the hair must lie exactly across these pieces.
They are then placed for 4-8 days, according to the thickness of the hide, in about 12 times their volume of a solution consisting of