A Comparative Study of Staining and Histochemical Reactions of the Components of Golgi Complex of the Fibroblasts in Vitro and the Ascites Tumor Cells

448 A Comparative Study of Staining and Histochemical Reactions of the Components of Golgi Complex of the Fibroblasts in vitro and the Ascites Tumor Cells Shunzo Takagi, Jin-ichi Kitada, Hideo Masuda and Masayuki Tagawa Departmentof Biology,University of OsakaPrefecture, Sakai,Japan ReceivedJune 27, 1961 It was previously shown with phase optics that the Golgi apparatus in living cells cultured in vitro, such as fibroblasts, corneal epithelial cells, myoblasts and white cells of the domestic fowl, consists of canalicules, which are usually seen as vacuoles in optical section, and of delicate filaments in a juxtanuclear area distinguishable from the rest of the cytoplasm (Takagi and Masuda 1956, Takagi 1958). Similar canalicules and filaments were also found to constitute the Golgi apparatus of frog lymphocytes (Takagi and Tagawa 1957). The walls of canalicules and the filaments are readily black ened by osmium tetroxide and the whole apparatus appears like a congeries of black strands. They are also stained vitally with Janus green and neutral red, and vitally and metachromatically with methylene blue, trimethylthionin and toluidine blue. The vital staining indicates that a canalicule is a dilated filament or often a dilated part of a filament. A number of electron microscopists have revealed that the Golgi complex consists of vacuoles, smooth membranes and small vesicles or granules, and the vacuoles are a dilatation of a double membrane, being characteristic of an actively secreting or absorbing cell (Haguenau and Bernhard 1955, Gatenby and Lufty 1956, Sjostrand 1956, Dalton and Felix 1956, 1957, Lacy 1957). The canalicules and the filaments observable in the living cells under the phase microscope may in all probability correspond to the vacuoles and the smooth membranes respectively in the electron micrographs, in which the latter often appear in the form of flattened vesicles and tubules. The other component of the Golgi complex, the small vesicles, being too small, are not to be seen under the phase microscope. As will be described later, the canalicular and the filamentous elements of the Golgi apparatus in cells vitally stained or stained after fixation both appear under the ordinary microscope as strands or filaments with various diameters. Hence, in the following lines, they will be referred to for convenience' sake as Golgi filaments. Our present knowledge on the staining and histochemical properties of the Golgi apparatus is based for the most part on studies of the structure identifiable as such by observing and comparing some standard Golgi prepara- 1961 Staining and Histochemical Reactions of Golgi Complex 449 tions with it. Elements of the Golgi complex directly observed in the living cell do not seem to have been the object of extensive study . Our present investigation aims at the study of whether the Golgi filaments, directly ob servable in the cultured fibroblasts and the mouse ascites tumor cells, are vitally stained, and after fixation are stained and whether they indicate any definite histochemical properties. The conspicuous result among many ob tained is that the Golgi filaments behaved themselves tinctorially very much like the mitochondria, although the former were distinct from the latter in some other features. The present investigation was made upon the basis of our observations of the Golgi apparatus in the two types of cells in living unstained condition, which will also be described. Materials and methods Fibroblasts cultivated from the heart of chick embryos and ascites tumor cells of the EM strain of the mouse were used. The former were derived from 8- to 9-day embryos and cultured in the medium of homologous em bryonal extract and plasma with the hanging drop method. The latter were supplied by the Zoological Institute of Hokkaido University. We observed in the previous study (Takagi and Masuda 1956) the Golgi apparatus of the fibroblasts in the living condition, after the addition of one per cent solution of osmium tetroxide and also in the Kolatchev preparation, and the organelle of the cells vitally stained with Janus green, neutral red and the other dyes. These observations were not, therefore, repeated in the present study. The ascites cells were observed with the use of a NDM phase objective (Tiyoda) in the ascites fluid mixed with an equal volume of a standard solu tion of powdered human serum. The Golgi filaments could be clearly dis tinguished from the mitochondria by this method. For the Kolatchev preparation, a drop of the ascites fluid was smeared on a coverglass, fixed in Champy and impregnated in one per cent osmium tetroxide. The direct action of osmium tetroxide on the Golgi filmaments of the ascites cells was examined under the phase microscope with the addition of a drop of one per cent solution to a drop of ascites fluid. Fibroblasts cultured on a coverglass and ascites cells smeared on a coverglass were fixed in Champy's fluid and stained with Altmann's acid fuchsin and picric acid with or without postchromation. In the vital staining of the tumor cells with Janus green, a drop of a 1:5,000 solution of the dye in Ringer was added to a nearly equal drop of the ascites fluid. The other vital dyes were applied also in a similar manner. The cells of two kinds on a coverglass were stained with Heidenhain's iron hematoxylin after being fixed in Champy's fluid and chromated in 2 per cent potassium bichromate. They were also stained with resorcin-fuchsin 450 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26 and counterstained with carmalum after fixation in 15 per cent formalin. The same materials were fixed in calcium-formol and were, after stain ing of the nucleus with carmalum or Delafield's hematoxylin, stained with sudan black. Fixed in the same manner, they were treated with trypsin and stained with Nile blue according to Tarao's procedure (1939) for the detec tion of masked lipid. This method was only applicable to the tumor cells, because the fibroblasts easily detached themselves from the coverglass during the treatment with trypsin. Baker's acid hematein test was employed for investigating phospholipid content of those two kinds of cells. Periodic acid-Schiff reaction after McManus was applied to the materials fixed in Gendre's fluid and digested with amylase. Alkaline phosphatase activity was tested according to Gomori's method. Results In phase microscopic observations of the fibroblasts in vitro, it was re peatedly seen that a clump of vacuoles and filaments, embedded in a ground substance which usually appears darker than the general cytoplasm, is always Fig. 1. Semi-diagram of the fibroblast (a) and the ascites tumor cell (b), showing the authors' interpretation of the structure of Golgi complex (in the figure, above the nucleus). Mitochondria and fat droplets are also shown. situated on one side of the nucleus. The vacuoles are 10-20 in number and usually 0.3-0.5 micron in diameter, and are in extreme cases as large as 1.0 micron. It was confirmed that the vacuoles represent optical sections of canalicules. The filaments run in various directions, but in elongated cells they are orientated almost parallel to the longer axis of the nucleus (Figs. la, 2). That the vacuolar and filamentous structures represent the elements of the Golgi complex of the electron microscopists is evident from the results of our previous study (Takagi and Masuda 1956) and from the facts to be 1956 Staining and Histochemical Reactions of Golgi Complex 451 described in the following pages. The ascites tumor cell shows under the phase microscope an area, lighter than its surroundings, of the cytoplasm on one side of the nucleus and usually at the center of the cell. In the area, filaments, extremely fine and gray, are seen running in various directions. They are thought to represent Golgi elements of the cell. There are no vacuoles in this area. The mito chondria in the form of short threads are orientated at random and are distributed in the peripheral cytoplasm, where a variable number of lipid droplets are found (Fig. 1b). In Kolatchev preparations of the fibroblasts, the Golgi apparatus consists of a clump of black threads of variable diameter, which are richly entangled, but do not form a real network (Takagi and Masuda 1956). In the ascites cells, it is likewise composed of thick black threads without anastomosis (Fig. 3). In some cells, the ground substance of the Golgi area is darkened with osmium and fine filaments are gray in it. In this case, the Golgi filaments are only slightly osmicated and their blurred contour is preserved. The mitochondria are usually not blackened in the same degree as the Golgi filaments. The lipid droplets are stained black. That the elements of the Golgi ap paratus are usually blackened with osmium Fig. 2. Living fibroblasts observed more easily than the mitochondria was with phase contrast. A cell to the left has a Golgi area on the left side shown only by the addition of one per cent of the nucleus, which is darker than solution of osmium tetroxide at room tem the surrounding general cytoplasm perature. Under the phase microscope, the and exhibits several Golgi vacuoles Golgi canalicules of the fibroblasts become within its confines. The other cell to the right has a similar Golgi area dark soon and appear like curved black in the upper right corner of the rods, which show a light unstained center nucleus. Mitochondria are shown in optical sections. It shows evidently that as threads, ranging from gray to the walls of the Golgi canalicules or vacuoles black. The cells contain also nu merous fat droplets. •~750 have reduced osmium tetroxide. The fila mentous Golgi elements also turn gray (Takagi and Masuda 1956).

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