Impregnation

IMPREGNATION (in histological technique) consists of the impregnation of objects, either in blocks or sections, with a solution of a metal salt, which is then reduced in certain areas of the tissue, causing these areas to appear colored black, brown, or another color depending on the quantity and properties of the reduced metal. In histological technique, silver salts (mainly silver nitrate AgNO₃), gold salts (mainly gold chloride AuCl₃+2H₂O), and osmic acid are used for the purposes of impregnation. A metal salt solution can be applied to either a fresh object, a fixed object, or finally, to sections prepared from it. The reduction of the salt is the most important phase of impregnation; it can be produced by the tissue itself without any other influence, or this process can be facilitated by introducing auxiliary factors under which the reduction processes occur significantly faster and more energetically (for example, light, acidification, heating), or finally, by resorting to reducing agents (formaldehyde, pyrogallic acid, etc.). Sometimes, upon completion of the reduction, the unreduced metal salt is removed by washing the preparation with a hyposulfite solution. If, with appropriate processing, only certain parts of the tissues are to be impregnated while the remaining tissue elements are to remain colorless, this is called a negative image. Conversely, positive images are sometimes obtained when all tissue elements are impregnated and only certain, very specific parts of them remain unstained. For the purpose of impregnation, silver nitrate was first proposed by Recklinghausen and His; it forms an easily reducible compound with the so-called cement substance of the epithelium and smooth muscles and with the ground substance of connective tissue, giving these parts a black or brown color; therefore, AgNO₃ impregnation is used as a reaction for detecting cement and ground substance, for which the methods of Ranvier, Hoyer, and others serve. In addition to this, silver is deposited in nerve cells (neurofibrils), axis cylinders, and, with specific processing and fixation, in glial elements (macro- and microglia), which is why impregnation is an indispensable method in the study of the nervous system and is widely applied. Among the methods, the following are most popular: the Golgi method and its modifications (Ramón y Cajal, Tishutkin, Kulchitsky, Bielschowsky). Since silver is also deposited in spirochetes, Levaditi proposed a method for the impregnation of spirochetes in tissues, which currently has many modifications. Gold chloride is reduced, just like osmic acid, in fats and parts of tissues rich in lipoids, especially myelin, staining them violet. In addition to this, gold reveals neurofibrils of nerve cells and the finest nerve threads and nerve endings (of the cornea of the eye, skin, terminal apparatuses). Gold chloride was introduced by Cohnheim (1866), and currently, his method has a multitude of modifications—Bastiani, Ranvier, Kolosov, Apáthy, and others. The methods are extremely capricious, and the conditions under which the best impregnation occurs are little known. Besides the use of gold or silver alone, combined treatment with both is used. The technique of impregnation was particularly finely developed in Spain by the school of Ramón y Cajal. The methods of Achúcarro, P. del Río-Hortega, Fafianas, and others, based on a purely photographic principle, allow for the revelation, with the help of impregnation in various sections of one and the same piece of tissue, of different elements, and also allow, if desired, to obtain a preparation that simulates the appearance of iron hematoxylin staining with additional counterstaining. See also Incrustation.