Vital Staining

VITAL STAINING, or in vivo staining, is the phenomenon of coloring tissues in a living organism through the introduction of various dyes. The definition of vital staining as the coloring of living tissues is not entirely correct, since in many cases it is extremely difficult to determine whether the coloring is present in 'living' elements to the full extent or in damaged, dying elements. In general, vital staining (V. s.) is distinguished from supravital (staining of dying elements) and postmortem staining. Various dyes introduced into the body for the purpose of vital staining must be non-toxic to the organism and must have the property of penetrating into tissues and remaining in them for a longer or shorter period of time. The results of vital staining vary completely depending on whether acidic or basic dyes are used. The results obtained with acidic dyes (see the main works of Bouffard, Goldmann, Kiyono, Schulemann, Chashin) depend less on their chemical composition than on the degree of dispersion and other physicochemical properties. Highly dispersed dyes do not produce coloring but diffusely impregnate the tissues and are quickly excreted from the organism. Therefore, for vital staining, colloidal or semi-colloidal coloring substances are used, for example, Trypan blue, Isamin blue, lithium carmine, and others. All these dyes are characterized by a negative charge of particles, slow diffusion, and insolubility in lipoids. After the introduction of acidic vital dyes into the organism, diffuse impregnation of the ground substance occurs, followed by accumulation of the dye in the protoplasm of certain cells of the organism in the form of granular deposits. In this way, only living cells are stained (the nuclei are not stained in the process). Dead cells are stained very diffusely, and their nuclei are also stained. Therefore, the method of vital staining is of great importance for distinguishing living cells from dead cells. Furthermore, with the help of vital staining with acidic dyes, it is possible to trace the process of distribution in the organism of many substances that are deposited in tissues in the same way as the mentioned dyes. These include: bile pigments, colloidal metals and other medicinal substances of a colloidal nature (for example, salvarsan), lipoids, and also, apparently, protein bodies, as well as various bacteria, various suspended particles of exogenous origin, some cellular elements and their breakdown products. The main place where the deposition of acidic vital dyes in granular form, as well as all the substances mentioned above, occurs is in the cells of the so-called reticulo-endothelial system (for details, see Reticulo-endothelial apparatus). It is particularly characteristic of vital staining of cells with acidic dyes that the constituent structural parts of the cell protoplasm are never stained in the process. The colored grains appearing in the cells are formed due to the precipitation of the dye from a dissolved state after it has penetrated into the cells. However, acidic dyes also impregnate to some extent certain preformed intracellular inclusions, especially of a protein nature. Vital staining in the proper sense of the word refers only to the deposition of dyes in cells in granular form. The mechanism of the formation of dye grains in cells is explained differently: according to some views, a gradual accumulation of the dye is noted in the cells within newly formed vacuoles, where there is a gradual decrease in the dispersion of the dye and finally its precipitation, in which the action of electrolytes is important (v. Mollendorff). Others attribute the main importance in the formation of intracellular dye grains to the phenomena of adsorption (Schulemann). Furthermore, there are indications that the dye always enters the cells in combination with plasma proteins, so the content of the latter in the blood is of great importance (de Haan, Zeeman). Then, in the process of vital staining with acidic as well as basic dyes, the concentration of H-ions in the tissues apparently plays some role (theories of Bethe, Rob.de, etc.). Besides the cells of the reticulo-endothelial system, granular deposits of acidic vital dyes are also formed in the epithelium of the convoluted tubules of the kidneys (through which, mainly, the excretion of these dyes takes place), and also, although not in all animals, in the cells of the liver. When large amounts of certain vital dyes (for example, Trypan blue) are introduced into the organism, it is also possible to obtain granular deposits of dye in other cellular elements, especially in the epithelium of endodermal origin (v. Mollendorff, Hesse, Glazunov, etc.), in various cells of the intermediate tissue, in the cells of many internal secretion organs, in the epithelium of the vascular plexuses of the brain, etc. Finally, it has been possible to obtain vital staining with acidic dyes of elements of the central nervous system (Rakhmanov, Behnsen, Mandelstam, etc.). In general, the result of vital staining, besides the properties of the dye, is also influenced by the method of introducing the dye, its dosage, and finally the condition of the tissues themselves, especially the degree of their blood supply. Interesting results were obtained with intravenous administration of non-toxic acidic dyes, when the rate of their disappearance from the blood (Okunev, Seyderhelm) and their further fate in the organism (Anichkov, Teplov, Kagan) were traced. With diffuse distribution of the dye in the tissues, the walls of the vessels are particularly sharply diffusely stained (Petrov). With subcutaneous and intraperitoneal administration of acidic vital dyes, it has also been possible to obtain, but more slowly, general staining of animals. In this case, the elements of the tissues at the site of dye administration are particularly sharply stained. There are indications that the reticular apparatus of the cells plays an important role in the deposition of dye grains in the cells (Golgi), since the appearance of grains always first occurs in the region of this apparatus (works of Nasonov, Khlopin, Yasnova). The previously expressed views that when stained with acidic dyes, the constituent parts of the cell protoplasm are stained, for example, mitochondria (Chashin, Steckelmacher), are now mostly abandoned. Besides the introduction of acidic vital dyes into the organism, the method of cultivating tissues in plasma containing these dyes is of great importance - especially Trypan blue (Hofmann, Maksimov, Vetteri, Khlopin). With this method, it is possible to observe vital staining of cells and study them in a living state. - Finally, the method of research by means of vital staining of living tissues directly under the microscope is important, as was achieved on some objects (lungs, urinary bladder, amphibian mesentery), when even strong immersion objectives can be used (works of Garmus, Vonwiller, Venslav). No vital staining of embryonic tissues is observed when acidic dyes are introduced into the maternal organism, since the placenta does not allow the dye to pass from the mother's blood. For staining embryonic tissues, the introduction of dyes into the amniotic cavity or, in birds, injection, for example, into the wall of the allantois, is used. The colloidal dye Congo red was specially proposed for the elective vital staining of amyloid (Bennhold); however, Trypan blue gives the same results (Gertzenberg). The application of vital dyes for vital staining of bones stands somewhat apart. Such dyes include derivatives of krappl, the coloring principle of which is alizarin. The staining of bones is based on the formation of a compound of alizarin with calcium, in which only young growing bones are stained (Lieberkuhn, Fischel, Gottlieb). Some products formed in the organism during pathological changes of Hb give the same bone staining as alizarin. This includes hematoporphyrin, which, when formed in excess in the organism, is deposited in the bones, staining the entire skeleton a sharply brown color (E. Fraenkel). A somewhat different method of vital staining is the so-called vital chemoscopy according to Karczag. This method is based on the ability of many dyes of the triphenylmethane group (for example, acid fuchsin, Liebgrim, Wasser blue) under the influence of certain effects (for example, light, heat, reducing substances, etc.) to pass into colorless carbine compounds. After the injection of these dyes, various tissues are examined, detecting the dye in them by the action of acid, which 'regenerates' the dye. Vital staining with basic dyes. Unlike acidic dyes, basic dyes stain pre-existing structural components of cells. It is assumed that in this case precipitation of the dye by the acidic colloids of the cells occurs (v. Mollendorff). Particularly sharp precipitation of the dye occurs when it is completely neutralized III; with an excess of dye or acidic colloid, various color shades of the stained elements are obtained, on which some methods for determining the concentration of H-ions in cells are based. Basic dyes usually penetrate into cells faster and precipitate sooner than acidic ones. By staining the structural components of cells, they give the same results on living and surviving objects. Therefore, they cannot be fully applied to distinguish living cells from dying cells to the same extent as acidic dyes. In general, for the occurrence of vital staining with basic dyes, the following conditions are important: the diffusion capacity of the dyes, on which the speed of staining depends, solubility in lipoids, the ability of the dye to be reduced, precipitability by acidic colloids, and finally, the content of H-ions in the tissues.

Particular importance was attached to the solubility of basic dyes in lipoids. The rate of onset of staining was largely dependent on this property, as it facilitates the penetration of the dye into the cell through the surface lipoid layer (Over-ton, Hober, Nierenstein). However, when it was discovered that dyes insoluble in lipoids also penetrate cells, the aforementioned view was strongly shaken. Along with the physical permeability of cells, the concept of their special physiological permeability was introduced (Höber). At present, the significance of lipoid components of the cell appears in a new light in terms of the possibility of accumulating dye at the lipoid-protoplasm boundary, due to the ability of dyes to reduce surface tension at this boundary (Okunev). The factor preventing the occurrence of V. o. with basic dyes is the property of the latter to be converted in tissues into colorless compounds by reduction. This property of dyes is also used to determine the sites of greatest oxygen consumption in tissues (Ehrlich, Unna and others). The components of cells that are stained by basic vital dyes are primarily various inclusions. In this respect, the action of basic dyes is partly similar to that of acid dyes. Furthermore, secretory granules, yolk plates, Nissl's granules in nerve cells, digestive vacuoles in protozoa, etc., are stained with basic dyes during life. The staining of cell granules with basic dyes observed by Arnold (Arnold) refers not to plastosomes, but mainly to secretory granules and inclusions. However, with certain basic dyes (Janus green), it is possible to obtain staining of plastosomes, especially in tissue cultures. Thus, basic vital dyes still mainly stain paraplastic substances, i.e., those that do not actively participate in cellular life. Among the most commonly used dyes for V. o. with basic dyes, Neutral-red, Nilblausulfat, Methylene-blue, Toluidin-blue, Thionin, Bismarckbrown, Crystal-violet, etc., should be mentioned. All these dyes are usually used in highly diluted solutions. Particularly good results are obtained with Neutral-red, Nilblausulfat, and Methylene-blue (numerous results of staining various animals with basic dyes, starting from protozoa, see the works of Nierenstein, Vonwiller, Loman, Stelanski, Fischel', Khlopin, and others). In many cases, it is very difficult to determine whether the staining with a basic dye has a vital or supravital character. Usually, supravital staining is more intense, and the structural elements of the nuclei are also stained. The staining of nerve fibers and endings according to Ehrlich (incorrectly called 'vital') is considered a representative of supravital staining (for more details on this method, see the works of Dogel and his school). Usually, the study of tissues with basic dyes is performed without fixation, on tissue spreads or on transparent membranes, especially in cold-blooded animals (see the works of Garmus, Vonwiller, Venslav). Furthermore, the method of growing tissues on plasma with the addition of basic dyes in very weak dilutions is also used (see the works of Vetter, R. Erdmann, Khlopin). Attempts to fix basic dyes in tissues have not given good results. Special mention should be made of the vital staining of fat, for which Sudan III dye is mainly used. The latter is introduced in solution in vegetable oil through the stomach or mixed as a powder with food. As a result, all fat depots of the body are stained. The mechanism of dye distribution and its penetration into fat depots is still little studied (see the works of Jakobsthal, M. V. Schmidt, and others).