A method of fundamental importance for following the nerve-paths through longer stretches was now introduced, namely, the method by series of consecutive sections, introduced by Benedikt Stilling. The necessity of cutting the brain and spinal cord into thin segments for the accurate investigation of their finer structure, even the older inves tigators recognized and suggested means by which to accomplish this end. As early as 1824, Rolando made thin cross-sections of hardened spinal cord with a razor and examined them with a hand-lens. But the segments cut by Rolando were not sufficiently thin to be used with higher amplification ; moreover, they were made without system. In 1836 Valentin examined microscopically the spinal cord of freshly-killed sheep and pigeons, by cutting the cord, under water, with a pointed two-bladed knife, into the thinnest possible lamellae and then carefully compressing the sections while being observed. In this manner Valentin studied the spinal cord layer by layer, from without inward, in longitudinal sections, and expressed the opinion, that for the correct understanding of the structure of the spinal cord examination by strata is the only proper way. Four years later, Hannover advanced along this line even farther than Valentin. He employed a brain and spinal cord hardened in chromic acid, which he cut into thin sections with a sharp knife, and examined the relations of these lamellae piece by piece.
Shortly after the appearance of Hannover's paper, the great doctor of Cassel, Benedikt Stilling, began in 1841 his investigations concerning the structure of the spinal cord. Stilling was the first to cut a spinal cord into many consecutive sections, as thin and transparent as possible, and then to study in each section the distribution of the white and gray substance. He traced progressively from section to section the changes in the picture, and finally, by reproducing the individual pictures, gained, at least to a certain degree, a clear conception of the internal structure of the cord. This method by series of consecutive sections, which Stilling designated as " investigation layer by layer," is even to-day the one most employed in the examination of the central nervous system. During the continual use of so productive a method, it was inevitable that the original technique of Stilling should undergo many alterations and improvements. The employ ment of the method was facilitated by better hardening of the organs. Already in 1832, Ludwig Jacobson recommended potassium chromate as a preservative for anatomical preparations. Hannover first put Jacobson's discovery to use for histological investigation. Later chromic acid was displaced from the technique by one of its salts. At any rate, no one has rendered greater service than Heinrich Muller by the introduction of potas sium bichromate, now so universally serviceable. From him came also the classic Miiller's fluid, which, indeed, even to-day is much used in its original composition. Later followed many new hardening reagents. One of these, formalin or formol, must be especially mentioned, since in recent years its many advantages have brought it into universal use. Formol was introduced in histological technique by Blum in 1893.
The method of consecutive sections was further facilitated by the introduction of the microtome, by which exact cutting and large regular sections are made possible, so that an entire brain may be laid into a series of thin sections without losing one. We may mark the sections in their proper sequence, determine in each the topography of the gray substance and of the fibre-tracts, and, by means of the series, from these isolated data construct a composite picture of the principles of construction of the part of the brain under discussion.
The application of Stilling's mode of investigation was materially facilitated by the methods of staining. For a long time Gerlach's carmine staining was dominant. An important advance was gained by Weigert's admirable hematoxylin-method. At present we have at our disposal a quite considerable number of different dyes, which may be used with advantage in investigating fibre-paths. But neither the Weigert stain, nor any other of the procedures so far recommended and used, is capable of solving the question, the answer to which has always been most sought for the correct understanding of the structure of the nervous system. Continually was asked : In what manner are the nerve-fibres related to the nerve-cells ? In what manner are the nerve-cells related to one another ? How do the nerve-fibres in the brain and spinal cord arise and how do they end ? In this connection, two methods were epoch-making—Ehrlich's methylene blue method and Golgi's silver-method. Ehrlich's procedure, which was introduced in 1886 and depends on the coloring of the living nerves by means of methylene blue, was quently improved by Retzius, Apathy, Bethe and others. Golgi's method is older. A number of years before, the Italian investigator had obtained preparations, in which the nerve-cells and their processes stood out with great sharpness as dark figures, by treating the brain-substance with the chromic acid salts and with silver nitrate. Golgi described his method as early as 1873, but at first his observations were little known. Not until the publication of an elaborate paper in 1886, did Golgi excite widespread attention and his results and methods become the starting point of an energetic examination of the central nervous system. For example, the Spanish savant, Ram6n y Cajal, was able, by the use of the Golgi method on embryos and young animals, to arrive at results that partly solved many of the dominant questions, or placed them in a new light. At first through the labors of this investigator, soon also through those of others, especially of Hiker, Lenhossek, van Gehuchten and Retzius, a clear picture took the place of the previous schemata. The most important findings of these researches are, that the nerve fibres are nothing more than extraordinarily long processes of the nerve-cells, that every nerve-fibre, from beginning to end, is to be regarded as a part of a single nerve-cell, and that every nerve-cell, with the nerve-fibres proceeding from it, represents an histo logical individuality or nervous unit. Waldeyer christened such anatomical unit, neurone, and therewith founded the neurone-theory.