endings seem satisfactorily stained. After placing the tissues on the slide, they are examined under the microscope (without covering with a coverglass) every two or three minutes, until such examination shows blue color in the neuraxes of the nerve-fibers and their terminations, or in the nerve-cells, if there be any in the tissues examined. Care should be taken not to miss the time when the staining has reached its full development, as the blue color usually fades again and only inferior preparations are obtained. Tissues thus stained may be fixed by one of two methods (or modifications of these methods), the selection of the method depending somewhat on the results desired. If it is desired to gain preparations giving the general course of nerves, the formation of nerve-plexuses, the relations of afferent and efferent nerves to the nerve-cells in ganglia, or the general arrangement of the terminal branches of nerve-fibers in nerve endorgans, the tissues are placed in a saturated aqueous solution of ammonium picrate (Dogiel) in which the blue color of the tissues is in a short time changed to a purplish color. In this solution the tissues remain for from twelve to twenty-four hours, and are then transferred to a mixture consisting of equal parts of a saturated aqueous solution of ammonium picrate and glycerin, in which they remain another twenty-four hours; they may, however, without detriment remain in the mixture several days. The tissues are then mounted in this ammonium picrate-glycerin mixture. If, on the other hand, it is desired to section tissues stained intra vitam in methylene-blue, the following method, slightly modified from that given by Bethe, is suggested. The following fixative is prepared: Ammonium molybdate, 1 gm.; distilled water, 10 c.c.; hydrochloric acid, I drop. The solution is prepared by grinding the ammonium molybdate to a fine powder, removing it to a flask, and adding the required quantity of water. The flask is now heated until the ammonium molybdate is entirely dissolved, when the hydrochloric acid is added. Before using this fixative it is necessary to cool it to 2°-5° C. It is, therefore, well to prepare it before the injection is made, and surround it with an ice mixture. In this fixative the tissues remain for from twelve to twenty-four hours. After the first six to eight hours it is not necessary to keep the fixative below ordinary room-temperature. After fixation the tissues are washed for an hour in distilled water. They are then hardened and dehydrated in absolute alcohol. It is advisable to hasten this step as much as possible, though not at the risk of imperfect dehydration. The tissues are then transferred to xylol and imbedded in paraffin, sectioned, fixed to the slide or cover-glass with albumin fixative, and may be double stained in alum-carmin or alum-cochineal. After staining in either of these stains, the sections are thoroughly dehydrated and cleared in oil of bergamot. The oil is washed off with xylol and the sections are mounted in Canada balsam. 312. In staining nerve-fibers with methylene-blue by local application of the stain to the tissues, the tissues to be studied are removed from an animal which has just been killed, divided in small pieces, and placed on a slide moistened with normal salt solution. A few drops of a TO to % solution of methylene-blue in normal salt solution are added from time to time-sufficient to keep the tissues moistened by the solution, but not enough to cover them. The preparations are examined from time to 404 Some time, under the microscope, to see whether the nerve elements are stained. In order to obviate the necessity for the low temperature of the vious method, Bethe (96) has recommended the following procedure: preAccording to the method of Smirnow and Dogiel, he first employs as a preliminary fixing agent a concentrated aqueous solution of ammonium picrate. In this he places the tissue, previously treated with methyleneblue, for from ten to fifteen minutes. objects are immersed in a mixture composed of ammonium molybdate Without further washing the larger (or sodium phosphomolybdate) 1 gm., distilled water 20 c.c., and pure hydrochloric acid 1 drop. The following mixtures may also be employed for the same purpose: ammonium molybdate (or sodium phosphomolybdate) 1 gm., distilled water 10 c.c., 2% solution of chromic acid Io c.c., and hydrochloric acid 1 drop; or, for very thin gross specimens or sections, ammonium molybdate (or sodium phosphomolybdate) 1 gm., distilled water, 10 c.c., 0.5% osmic acid 10 c.c., and hydrochloric acid I drop. Small objects are permitted to remain no longer than from three quarters of an hour to one hour in either of the first two mixtures, and not more than from four to twelve hours in the third. specimens are washed with water, carried over into alcohol, then into xylol, After fixing, the and finally imbedded in paraffin. Subsequent staining with alum-carmin, alum-cochineal, or one of the neutral anilin dyes gives good results. 313. A very promising method recommended by Meyer (95) consists in injecting subcutaneously about 20 c.c. of normal salt solution containing from 1% to 4% of methylene-blue into a young rabbit, and repeating the operation in one to two hours. Within the next two hours the animal usually dies and the central nervous organs are then removed and small pieces fixed according to Bethe's method. 314. Apathy (97) demonstrates the fibrillar elements of the nervous system in invertebrates and vertebrates by means of his gold method. Fresh tissue may be used, or tissue already fixed. membranes are placed for at least two hours in a 1% solution of yellow In the first case thin chlorid of gold in the dark, then carried over without washing into a 1% solution of formic acid (sp. gr. 1.223), and finally exposed for from six to eight hours to the light (the formic acid may have to be changed). These specimens are best mounted directly in syrup of acacia or in concentrated glycerin. In his second method Apáthy fixes vertebrate tissues for twenty-four hours in sublimate-osmic acid (1 vol. saturated solution of corrosive sublimate in 0.5% sodium chlorid solution combined with 1 vol. 1% osmic acid solution), washes repeatedly in water, and places for twelve hours in an aqueous iodo-iodid of potassium solution (potassium iodid 1% and iodin 0.5%). The further treatment is the same as after ordinary corrosive sublimate fixation. Finally, the specimens are imbedded in paraffin with the aid of chloroform, cut, and mounted by the water method. The whole process, up to the point of imbedding in paraffin, is carried out in the dark. The sections are then passed through chloro form and alcohol into water, where they are allowed to remain for at least six hours; or they may be washed in water, placed for one minute in 1% formic acid, again washed in water, immersed for twenty-four hours in a 1% solution of gold chlorid, rinsed in water, and finally placed in a 1% formic acid solution and exposed to the light. For the latter purpose glass tubes are employed in which the slides are placed obliquely, with the sections downward. A uniform illumination of the section with "as intense a light and low a temperature" as possible are conditions indispensable to the attainment of successful results. The sections are then again washed in water and mounted in glycerin or syrup of acacia, or in Canada balsam after being dehydrated. Thin membranes are stretched upon small frames of linden wood especially prepared for this purpose. Their further treatment is then the same as that of sections fixed to the slide. If successful, the nerve-fibrils appear dark violet to black. The large ganglia in the spinal cord of lophius, the calf, etc., are especially recommended as appropriate vertebrate material. Bethe (1900) has recommended the following method for staining neurofibrils and Golgi-nets in the central nervous system of vertebrates: The perfectly fresh tissue is cut in thin lamellæ, varying in thickness from 4 to 10 mm. These are placed on pieces of filter-paper and then in 3 to 7.5% nitric acid, in which they remain twenty-four hours. From the hardening fluid the pieces of tissue are transferred into 96% alcohol, where they remain for from twelve to twenty-four hours. They are then placed in a solution of ammonium-alcohol,-ammonium (sp. gr. 0.95 to 0.96), I part; distilled water, 3 parts; 96% alcohol, 8 parts,in which they remain for from twelve to twenty-four hours. The temperature of this solution should not exceed 20° C. The tissues are then placed for from six to twelve hours in 96% alcohol, and next in a hydrochloric acid-alcohol solution,-concentrated hydrochloric acid (sp. gr. 1.18-37%), 1 part; distilled water, 3 parts; and 96% alcohol, 8 to 12 parts, in which they remain for several hours. The temperature of this solution should not exceed 20° C. The tissues are then again placed in 96% alcohol for from ten to twenty-four hours, and next in distilled water for from two to six hours. The tissues are now placed for twenty-four hours in a 4% aqueous solution of ammonium molybdate. (This solution should be kept at a temperature varying from 10° to 15° C., if it is desired to stain the neurofibrils; or at a temperature varying from 10° to 30° C., if it is desired to bring out the Golgi-nets.) After the ammonium molybdate treatment, the tissues are rinsed in distilled water, placed in 96% alcohol for from ten to twenty-four hours, then in absolute alcohol for a like period, cleared in xylol or toluol, and imbedded in paraffin. Sections having a thickness of 10 μ are now cut and fixed to slides with Mayer's albumin-glycerin. Since the various solutions used in the fixation and further treatment of the tissues do not act with the same intensity on all parts of the piece of tissue to be studied, and since the differentiation and staining depend on a relative proportion (not yet fully determined) of the mordant (ammonium molybdate) and the stain in a given section, it is advised by Bethe to cut large numbers of sections and fix to each slide about three sections from different parts of the series. After fixation of the sections to the slide the paraffin is removed with xylol; and they are then carried through absolute alcohol into distilled water, in which, however, the sections remain only long enough to re move the alcohol. The slides (with the sections fixed to them) are then taken from the water and rinsed with distilled water from a water-bottle. The slide is then wiped dry on its under surface and edges with a clean cloth, and about 1 c.c. to 1.5 c.c. of distilled water placed on the slide over the sections. The slides are now placed in a warm oven with a temperature of 55° C. to 60° C. for a period of time varying from two to ten minutes. No definite time can here be given; sections from each block of tissue need to be tested until the right stay in the warm oven is ascertained. The slides are then taken from the warm oven and rinsed two or three times in distilled water and again dried as previously directed. They are then covered with the following staining solution and again placed in the warm oven for about ten minutes: toluidin-blue, I part; distilled water, 3000 parts. The stain is washed off with distilled water and the sections are placed in 96% alcohol until no more stain is given off-usually for from three-fourths to two minutes. They are then dehydrated in absolute alcohol, passed through xylol twice, and mounted in xylol balsam. For a fuller discussion of this method the reader is referred to Bethe's account in "Zeitsch. f. Wissensch. Mikrosk.," vol. XVII, 1900. 315. For staining neuroglia Weigert (95) has recommended a method, from which we give the following: A solution is made consisting of 5% neutral acetate of copper, 5% ordinary acetic acid, and 2.5% chrome-alum in water. The chrome-alum and water are first boiled together, the acetic acid then added, and finally the finely pulverized neutral copper acetate, after which the mixture is thoroughly stirred and allowed to cool. To this solution 10% formalin may be added. Objects not over 0.5 cm. in diameter are placed in this fluid for eight days, the mixture being changed at the end of a few days. By this means the pieces of tissue are at the same time fixed and prepared for subsequent staining by the action of the mordant. If separation of the two processes be desired, the specimens are fixed for about four days in a 10% formalin solution (which is changed in a few days), and then placed in the chrome-alum mixture without the addition of formalin, Specimens thus fixed may be preserved for years without disadvantage, and may then be subjected to further treatment by other methods, Golgi's for instance. Washing with water, dehydration in alcohol, and imbedding in celloidin. are the next steps. The sections are then placed for about ten minutes in a 0.33% solution of potassium permanganate, washed by pouring water over them, and placed in the reducing fluid (5% chromogen and 5% formic acid of a specific gravity of 1.20; then filter carefully, and add 10 c.c. of a 10% solution of sodium sulphite to 90 c.c. of the fluid). The sections, rendered brown by the potassium permanganate, readily decolorize in a few minutes, but it is better to leave them for from two to four hours in the solution. If it be desirable to decolorize entirely the connective tissue, no further steps need be taken preliminary to staining; if not, the reducing fluid is poured off and the sections are rinsed twice in water and then placed in an ordinary saturated solution of chromogen (5% chromogen in distilled water, carefully filtered). The sections are left in this solution overnight, and the longer they remain in it, the more marked will be the contrast, as far as stain is concerned, between the connective and nervous tissues; then water is again twice poured upon the sections and they are ready for staining. This process consists in a modified fibrin stain (vid. Technic). The iodo-iodid of potassium solution is the same (saturated solution of iodin in a 5% iodid of potassium solution). Instead of the customary gentian-violet solution, a hot saturated alcoholic (70% to 80% alcohol) solution of methyl-violet is made, and, after cooling, the clear portion decanted off; to every 100 c.c. of this fluid 5 c.c. of a 5% aqueous solution of oxalic acid is added. The staining takes place in a very short time. The sections are then rinsed and normal salt solution and the iodo-iodid of potassium solution poured over them (5% iodid of potassium solution saturated with iodin), and washed off with water and dried with filter-paper and decolorized in the anilin oil-xylol solution in the proportion of 1:1. The reactions are rapid, and the thickness of the section should not exceed 20 μ. This method is best adapted to the central nervous system of the human adult ; it has as yet not been sufficiently tested for other vertebrates. VIII. THE EYE. A. GENERAL STRUCTURE. THE organ of vision consists of the eyeball, or bulbus oculi, and the entering optic nerve. In the eyeball we distinguish three tunics: (1) a dense external coat, the tunica fibrosa or externa, which may be regarded as a continuation of the dura mater, consisting of an anterior transparent structure, called the cornea, and the remaining portion, known as the tunica sclerotica, or, briefly, the sclera; (2) within the tunica fibrosa a vascular tunic, the tunica vasculosa or media, subdivided into the choroid, ciliary body, and iris; (3) an inner coat, the tunica interna, which consists of two layers, the inner being the retina; the outer, the pigment membrane. The latter lines the internal surface of the tunica vasculosa throughout. Within the eyeball are the aqueous humor, the lens, and the vitreous body. The lens is attached to the ciliary body by a special accessory apparatus-the zonula ciliaris. These two structures—the lens and its fixation apparatus-divide the cavity of the eyeball into two principal chambers, the one containing the aqueous humor and the other the vitreous. The former is further subdivided by the iris into an anterior and a posterior chamber. During life the latter is only a narrow capillary cleft. B. DEVELOPMENT OF THE EYE. In man the eyes begin to develop during the fourth week of embryonic life, and at first consist of a pair of ventrolateral diverticula, projecting from the anterior brain vesicle. These evaginations gradually push outward toward the ectoderm, and are then known. as the primary optic vesicles. The slender commissural segments |