247 STUDIES OF MITOCHONDRIAL STAINING WITH PINACYANOLE, EMPLOYI}.TG YOSHIDA ASCITtsS SARCOMA CEI-L KryosARU TexrrAwA, Kyoreno Aen, Krsuro Kero, Tnnuo Yosnloa AND Kyorcnr MesurANr Department of Internal Meclictne, I(omatsujima RerI Cross Hospital (Chief : Dr. Riyoharu Takikau)a) 7st Departntent of Internal Medicine, Nagoya Uniuersity School of Medicine (Director : Prof . Susumu Hibino) Because of potent activities of respiratory enzymes found in isolated mito- chondria, morphological changes in mitochondria have again attracted attention as indicative of cell's functional potentialities. Mitochondria in the cell can be visualized by employing, 1) Altmann's stain- ing method or Heidenhain's iron hematoxylin stain on fixed preparations, 2) the supravital staining method using Janus green, and recently 3) the supravital observation by means of the phase contrast microscope. Among these, the supravital method with Janus green is widely employed because of its simplicity and high specificity. But this Janus green method is not free from faults : namely difficulty in differentiating the types of cells and quick fading of the stained mitochondria. In 1936 Hetheringtonl) introduced a dyestuff named pinacyanole into the su- pravital staining method of mitochondria, and this method has been investigated by J. L. Schwind,2) showing that nuclei are stained supravitally and the types of cells are easily differentiated, stainability of neutral red vacuoleg is not dis- turbed and the colored mitochondria do not fade away for several hours. This pinacyanole (Consolidated Midland Corporation) and vital neutral red have been obtained lately, and we are discussing the usefulness of the former dyestuff in the study of mitochondria, comparing it with the above-mentioned various mitochondrial methods, and the nature of its staining mechanism. Furthermore, we report here in the second chapter, a new, simple and ef- fective mitochondrial staining method on fixed smear preparations of cells. I. tns suPRAVTTAL METHoD Material and method Yoshida ascites sarcoma cells were employed. The reason for this choice of material was that its huge cell body, its long and fine mitochondria, and its brilliant rosette of neutral red vacuoles make discussion of the details of the cytological structure easy. Received for publication November 25, L953. 248 K. TAI(IKAIVA ET AL. In addition, the influence of the supravital staining procedure upon the movement of neutrophile leukocytes was investigated. Altmann's original method was applied to the Yoshida ascites sarcoma cell smears fixed in Champy's fluid. Janus green and neutral red supravital staining r.vas done with the "isotonic saline solution method." Chiyoda's phase contrast microscope with dark medium DM and bright medium BM objectives was employed. Pinacyanole solution in absolute alcohol (500-1 000 x ) (or together with vital neutral red) was dropped on a slide, left to dry, and a drop of the cell suspen- sion (Yoshida sarcoma ascites) was placed on this dye film over the slide-glass, then covered with a cover-glass (the so called "dye film method"). Obseruations Findings with Giemsa's stain: The Yoshida ascites sarcoma cell is a huge, clearly defined, round, oval or kidney shaped cell which has a large round nu- cleus providing a delicate homogenous nucleal network and a few large oval or comma shaped nucleoli. Its cytoplasm reveals an even strongly basophile granu- lar appearance. It happens frequently that a rosette of azur granules, coarse and irregular in size, is seen in the bay of the nucleus r.vhich is named the achromatic circle or the hof. This is called the azur rosette. Altmann's staining: Fine rod-shaped or filamentous mitochondria are stained in great numbers around the bay of the nucleus and in lesser numbers surround- ing them, in the cytoplasm. But there are seen a number of large and small irregular shaped granules which do not appear as mitochondria. Supravital stain by means of Janus green and neutral red: There is a rosette of neutral red vacuoles in the bay of the nucleus. This is called the neutral red rosette. Surrounding this, many mitochondria, rod shaped or fila- mentous, which are stained greenish blue by Janus green are detected. The nucleus is not colored. Finding with the phase contrast microscopy: The nucleus appears bright and the nucleolus is dark with the use of DM objective Chiyoda. A circular .arrangement of bright granules is visible around the bay of the nucleus which is thought to be composed of an azlur granule rosette and of some fat granules. By BM objective, dark mitochondria are clearly distinguished. Fat granules in the cytoplasm appear bright by DM objective, as by BM. Supravital stain with pinacyanole and vital neutral red: Bluish stained rods and filaments are seen distinctly gathered around the area adjacent to the nu- .cleus. But Iarge and small irregular shaped granuies, also stained bluish, are seen; and bright particles (droplets) here and there in the cytoplasm become stained light bluish as time elapses. At first, the nucleus is stained diffusely and the nucleolus alone is plainly visible. Then frequently the nuclear network gradually becomes visible. Rods and filaments remain stained at this time, but the cytoplasmic structure does not color diffusely. Stained with the vital neutral red together, the neutral red rosette is also seen, but this method is accompanied by some difficulty. N{ITOCI{ONDRIAI- STAINING i/ViTI{ PINACYANOLE, I1^MPLOYI}{G YOSI{IDA ASCITES SARCOMA CELL 249 Discussion Altmann thought granules stained with his anilin water acid fuchsin method were most important in cell existence and named them "Bioblasten." Various names were adopted by different authors, but now this body is called mito- chondria. However, not all bodies which can be stained by this method are mitochondria. Identification of mitochondria is used to be made by its figure and location, and by comparing with other mitochondrial stainings. By supravital staining method using Janus green, particles colored by a solu- tion of Janus green diluted with more than 10 000 times physiological saline can be designated as mitochondria. (This is the "saline solution method.") The so-called oodye film method," in which a drop of the cell suspension is placed on a dye film, seems to be convenient for observation of the ameboid movement of leukocytes, but the dye concentration which acts on the cell mem- brane becomes irregular. It often differs markedly in dye concentration between the peripherai and the central portion in the preparation of this dye film methed. In phase contrast microscopy, the bright and the dark represent a difference in optical density of the visible cytoplasmic details. It is said that mitochondria are of the highest optical density, burt some cellular components of similar den- sity may be mistaken for mitochondria. Now, we shall attempt to discuss whether the particles stained with the pinacyanole are mitochondria or not, and, then, if so, what merits or demerits are found in this staining compared with the Janus green method. l. It wouid seem reasonable that the cell to be tested should be brought into the isotonic saline solution of the pinacyanole in order to prescribe a uniform dye concentration. But pinacyanole (Consolidated Midland Corporation) is so insoluble in isotonic saline that the "isotonic saline solution method" can not be applied. This is the only grave fault of this dyestuff, and therefore the "dye film method" must be adopted. Pinacyanole is quite insoluble in water, in alkali (sodium hydroxide, am- monia and sodium carbonate in aqueous solutions) and in saline solution. It is soluble in mineral acids (muriatic acid, sulphuric acid and nitric acid) but de- coloration takes place. Pinacyanole is easily soluble in alcohol and in warmed fats. So this dyestuff must be a color base. The dyestuff sudan III is also in- soiuble in water and soluble in alcohol and in fats. Therefore, Demel's3) supra- vital staining method for fats (sudan III film method) by which neutral fat droplets in the cell are stained specifically, as confirmed previously by us,a) is comparable with this supravitai staining method by pinacyanole dye film in its staining mechanisms. Demel's supravital method consists of these steps : Sudan III is dissolved in absolute alcohol, boiled, filtered and dropped on a glass-slide and then dried. 'fhis is the sudan III dye film and on it is placed a drop of a cell suspension to be tested, then a cover-glass is placed over it. In ascites five days after a transplantation of Yoshida sarcoma, bright droplets particles in the sarcoma cell become stained a yellowish'orange color. This droplet seerns identical with the so-calied neutral red staining refractile particle in Iymphocytes.s) We cali this "Demel's granule" (neutral fat). 250 K. TAKII(AWA ET AL. If a pinacyanole dye film is used in the supravital staining of Yoshida sar- coma cell, droplets which are stained orange by Dernel's dye film method ("Demel's granule"), can be colored bluish with pinacyanole at this time. And, as a matter of course, rods and filaments are stained blue simultaneously. Upon comparison with Altmann's staining, supravital Janus green staining and examination by pirase contrast microscopy, it is seen without doubt that rods and filaments stained supravitally by the "pinacyanole dye film method" are mitochondria. But because the dye concentration can not be maintained homogeneously, confusion may arise from the fact that lipid granules other than mitochondria may also be stained. Pinacyanole is soluble in serum and ascites fluid. When this pinacyanole- serum is mixed with Yoshida sarcoma ascites (our pinacyanole-serum method), mitochondria of the sarcoma cell is also stained well. Therefore the staining mechanism of the supravital mitochondrial staining in the pinacyanole dye film method must be as following: the pinacyanoie dis- solves out of the dye film over the glass-slide through the lipid component in the ascites (the cell suspension) and melts in the cell membrane of Yoshida sarcoma cell.