Natural injection of Medusæ may be effected without injuring the vessels, with an opening at the side remote from it. The medusa must be placed in a glass vessel, with the bell downwards, and a bell-jar ending in a narrow tube above is placed over it and made air-tight; the medusa is then covered with the injection-mass, the air in the glass is exhausted, and as the sea-water runs out by slits in the lower side of the annular canal, the coloured fluid runs in. In the case of leeches and large species of earthworms, the natural injection is made from the ventral sinus. In all cases a glass tube is used, with a finely drawn-out point. The injection is complete when the injection issues from the counter-opening. Besides the animals mentioned, large caterpillars, beetles, and larvæ of various kinds are favourable objects for injection; the glass cannula being introduced into the posterior end of the dorsal vessel, and the counter-opening made in the ventral vessel, and vice versâ.
Staining Living Protoplasm with Bismarck Brown.—Henneguy having treated Paramœcium aurelia with an aqueous solution of aniline brown (known as “Bismarck Brown”), found that they assumed an intense yellow-brown colour. The colour first appears in the vacuoles of the protoplasm, and then in the protoplasm itself, the nucleus generally remaining colourless, and becoming more visible than in the normal state. If a yellow-tinted paramœcium be compressed so as to cause a small quantity of the protoplasm to exude, it is seen that it really is the protoplasmic substance which becomes coloured. All the Infusoria may be stained with Bismarck brown, but no other aniline colour employed exhibits the same property—they merely stain the Infusoria after death, and are in fact poisonous. Living protoplasm does not as a rule absorb colouring matters, and as Infusoria are chiefly composed of protoplasm, attempts have been made to ascertain whether protoplasm in general, of animal or vegetable origin, behaved in the same way in the presence of aniline brown. A tolerably strong solution of Bismarck brown was therefore injected under the skin of the back of several frogs. After some hours the tissues became uniformly tinted a deep yellow; the muscular substance especially had a very marked yellow tint. The frogs did not appear in the least incommoded. Small fry of trout placed in a solution stained rapidly and continued to swim about. Finally, a guinea-pig, under whose skin some powder of Bismarck brown had been introduced, soon presented a yellow staining of the buccal and anal mucous membranes and of the skin. Seeds of cress sown on cotton soaked with a concentrated solution of the Bismarck brown sprouted, and the young plants were strongly stained brown; but on crushing the tissues and examining them under the microscope, it was ascertained that the protoplasm of the cells was very feebly coloured: the vessels, on the contrary, showed a deep brown stain up to their termination of the leaf. The mycelium of a mould developed in a solution of Bismarck brown was clearly stained after having been washed in water, whilst it is known that the mycelium, which frequently forms in coloured solutions, picro-carmine, hæmatoxylin, &c., remained perfectly colourless. Other aniline colours injected under the skin of frogs stained the connective tissue as deeply as did the Bismarck brown; but the striæ of muscle remained colourless. We may conclude, then, that Bismarck brown possesses the quality of colouring living protoplasm both in plants and in animals.
Cutting, Grinding, and Mounting Hard Structures.
Take the femur of cat, or rabbit, remove as much of the muscle as possible and macerate it in water until quite clean; on removal hang it up to dry. With a fine saw make transverse and longitudinal sections. File the section down until flat, and smooth. Take some Canada balsam, place a piece on a square of glass and warm gently over a lamp until the balsam is plastic enough to allow of the section being pressed into it, and set it aside to consolidate. Take a hone (“Water-of-Ayr” stone), moisten it with water, and rub one side of the section upon it until quite smooth, then place the glass slip, with the section still attached, into methylated spirit, and in a very short time the section will be separated; wash it and remount it on the reverse side, and proceed to rub it down on the hone until it appears to be thin enough for mounting. Polish both sides on a polishing strop with Tripoli powder, and mount in Canada balsam.
Fig. 244.—Small Lathe for cutting and polishing Sections of Teeth.
Teeth.—The enamel of the teeth is a much harder structure than that of bone, consequently it is found necessary to have recourse to a cutting machine. Hand machines have been introduced for this purpose, but the small lathe described in the earlier editions of my book has in no way been superseded by later cutting machines. [Fig. 244] represents the small lathe used for cutting and polishing every kind of hard substance. With regard to the teeth, two sections should be made perpendicular to one another through the middle of the crown and fang of the tooth from before backwards, and from right to left, which will show the peculiar structure of the enamel. The section must be cemented to the carrier of the stock of the lathe, or to the metal plate a, and kept in position by the steel holder b; the wheel being set in motion by the first treadle. The embedding materials in use are either gum-shellac or Canada balsam. The former is more generally employed by the lapidary and grinder of lenses than the latter. As the enamel is liable to fracture under the saw, it will be necessary to lessen the friction by dripping water on the saw as it is made to revolve. Thick sections can be quickly ground down against the corrondum wheel. The final polishing of the section may be done on the lathe, or by rubbing the flattened surface with water upon a “Water-of-Ayr” stone, and ultimately set up in Canada balsam, which must not be too fluid, or it will penetrate the lacunæ and canaliculi, fill up the interspaces, and cause them to become quite invisible. As the flatness of the polishing surfaces is a matter of importance, the stones themselves should be tested from time to time, and when found to present an uneven surface must be rubbed down on a granite stone with fine sand, or on a lead plate with emery powder. If it is decided to use Canada balsam as the embedding material, this must be prepared in the following manner:—The section of tooth or bone must be attached to a slip of well-annealed glass by hardened Canada balsam, and its adhesion effectually secured by placing the slide on the cover of a water bath, or in the hot-chamber ([Fig. 256]), when the balsam, a thick drop of which should be used, will spread out by liquefaction. The slide should then be removed and allowed to cool in order that the hardness of the balsam may be tested. If too soft, as indicated by its readily yielding to the pressure of the thumb-nail, the heating process must be repeated, care being taken not to cause it to boil and form bubbles; if too hard, which will be shown by its chipping, it must be remelted and diluted with fluid balsam, and then set aside as before. When it is found to be of the right consistence, the section must be laid upon its surface with the polished side downwards; the slip of glass is next to be gradually warmed until the balsam is softened, care being taken to avoid the formation of bubbles, then press the section gently down with a needle upon the liquefied balsam, the pressure being just applied on one side rather than over the whole surface, so as to drive the superfluous balsam towards the opposite side; finally, an equable pressure over the whole will secure a perfect attachment of the section without air bubbles. If, however, these should present themselves in drying, and they cannot otherwise be expelled by pressure, it will be found better to take the section off and relay it as before. The thickness of the layer of balsam may be reduced by rubbing it down before applying the glass-cover.
Rock Sections.—Small pieces of rock may be ground down by the aid of the lathe, or on a zinc plate, with emery powder and water, until one side is rendered smooth and flat. Then fasten the polished side of the section to a square of glass on the metal holder of the lathe, with dried Canada balsam, as directed for bone, and allow it time to become consolidated. When moderately thin take a piece of plate-glass and some fine emery or putty-powder and rub the section down as thin as possible. When found to be thin enough wash it well in water, and put it aside to dry, or warm it over a spirit-lamp, and with a needle push the section off the glass into a watch-glass of benzole or turpentine, and allow it to soak until all the balsam is dissolved out. Wash again in turpentine, and mount in Canada balsam, with or without a cover-glass. Sections of echinus spines, shells, stones of fruits, &c., are prepared in the same way as those of bones and teeth; but when the grinding is finished, the sections must be passed through alcohol into oil of cloves, after which they should be mounted in Canada balsam. If tolerably thin, sections of these substances can be cut in the lathe; in the first instance, there will be no actual occasion to attach them to glass at all, except for the purpose of obtaining a hold upon the specimen for polishing, but the surface thus attached must afterwards be completely removed in order to bring into view a stratum which the Canada balsam may not have penetrated.
With regard to smaller bodies, these can scarcely be treated in any other way than by attaching a number of them to slips of glass at once, and in such a way as to make them mutually support each other. Thus in making horizontal and vertical sections of foraminifera, it would be impossible to slice them through unless they were laid close together in a bed of hardened Canada balsam, and first grinding away one side and then turning and rubbing down the other. My friend, Dr. Wallich, many years ago communicated to me the ingenious plan adopted by himself when mounting and turning a number of these minute objects together. The specimens being cemented with Canada balsam, in the first instance, to a thin film of mica, and then attached to a glass slide by the same means, when ground down to the thinness desired, the slide must be gradually heated just sufficiently to allow of the detachment of the mica-film and the specimen it carries; a clean slide with a thin layer of hardened balsam having been prepared, the mica-film is transferred to it with the ground surface downward. Its adhesion by drying having been complete, the grinding and polishing should be proceeded with; and as the mica-film will yield to the stone without any difficulty, the specimen now reversed in position may be further reduced to the requisite thickness for mounting as a permanent object.
Staining and Mounting Vegetable Tissues.—Bacteria I propose to treat of in a separate section. Vegetable tissues generally will first receive attention, and their differentiation is based on the employment of delicate gradations of colour stains. The more striking results are obtained by Multiple Staining, while the cell contents are rendered more palpable. On this account colouring media have been divided into nuclear, plasmic, and specific. The first are chiefly valued in proportion as they prove to have a selective affinity for the nuclei of cells, and leaving the protoplasm comparatively unstained. Such stains are needful for fresh and young tissues. On the other hand, plasmic stains colour tissue uniformly, and are used to give a ground colour by way of contrast; and specific stains are chiefly employed to distinguish certain elementary structures from the mass of cellulose which forms the basis of vegetable tissue, and which is also met with to a slight extent in animal membranes.