STUDIES of MITOCHONDRIAL STAINING with PINACYANOLE, EMPLOYI}.TG YOSHIDA Ascittss SARCOMA CEI-L

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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 mitochondria, 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 staining 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 supravital 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 effective 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 suspension (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 nucleus 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 surrounding 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 filamentous, 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 mitochondria. 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 solution 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 membrane 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 density 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) supravital 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 sarcoma 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. This dyestuff is brought into the cytoplasm and is condensed into mitochondria which has been found to represent a metabolic center.6) Be- cause mitochondria are thought to consist mainly of lipoid and protein sub- strates,6) the pinacyanole is fixed on mitochondria as soo11 as stains it. 2. It is natural that using the same "dye film method," staining rates between water soluble and insoluble dyestuffs differ remarkably. A cell suspension over a water soluble dye film soon becomes saturated and the cell in it is. easily overstained. Because water-insoluble dyestuff such as pinacyanole dis- solves according to the lipid content in the cell suspension medium, the dye density in it is almost ahvays small. Even if a thicker dye film is employed, the coloration in the cell components does not differ sharply, and dye crystals. remaining oyer the slide-glass are detected plentifully at this time. This may be one reason why all the cell components are not colored diffusely by pinacyanole even though several hours be allowed for staining. 3. The reason the stained mitochondria does not fade, may be that the water' insoluble dye can hardly be reduced by enzymes in the cytoplasm. 4. On the supravital nucleal staining: When the blood was stained supravitally by the "pinacyanole dye film method," it has been found that the nucleus of neutrophiles become stained at first diffusely, and Iater its network was clearly seen. And yet neutrophiles move activeiy, therefore the staining of the nucleus is not a postmortal stain but must be a supravital nucieal stain. If so, the pinacyanole may be a peculiarT) dyestuff which reveals not only the nucleal shape but also its network supravitally. Article 1 above is a demerit, and articles 2, 3 and 4 are merits of this pinacyanole color base. Accounts of supravitai staining method with water insoluble dyestuff are rare in literature and among them only the Demel's method with the sudan III is of importance. The mechanism of the supravital stainings in this Demel's method and the pinacyanole method stated here, is characterized by the peculi- arity of the dye film method in itself and by the insolubility of these dyestuffs IVIIIIOCFIONDRIAL STAINING WITII PINACYANOLE, 2s1 EMPLOYING YOSFIIDA ASCITES SARCOMA CELL

in water. But in Demel's method, mitochondria cannot be stained supravitally and this is due to the fact that the suclan III is not a color base but an "indifferent dyestuff."sr Summary 1. It has been proved that mitochondria can be stained by means of the supravital staining method with pinacyanole. 2. Pinacyanole is insoluble in water, easily soluble in alcohol and soluble in warmed fats. So it may be a color base (not a salt). It is a grave fault that the "isotonic saline solution method" can not be employed in this supravital staining. Therefore the "dye film method" must be adopted. Because this water insoluble pinacyanole film over the slide-glass can dissolve out only through the fatty component in the celi suspension medium (Yoshida sarcoma ascites), concentrations of pinacyanole in the medium which act upon the cell membrane may Iose their homogeneity. Thus confusion may result from the ,coloring of fatty granules other than mitochondria. But if the serum saturated with pinacyanol is used to the supravital staining procedure (our pinacyanole- serum method), this confusion is not inevitable. 3. The supravitally stained mitochondria does not fade even after several hours. 4. Pinacyanole can hardly overstain all the cytoplasm diffusively. 5. Because the nucleus can be stained without interfering with the ameboid movement of the neutrophile leukocyte, this may be called supravital nucleal staining. This staining therefore is convenient for distinguishing the types of cells. Article 2 above is a demerit and 3,4and5 aremeritsconcerningthenatqre of this color base. And this supravital method, together with Demel's, shol characteristics of the dye film method itself and of these dyestuffs insoluble in w-ater. But with Demel's method mitochondria is not stained supravitally and this is due to the fact that the sudan III is an "indifferent dyestuff" insteadof .a color base. 6. The so called neutral red staining refractile particle seems to be identical with Demel's granule which can be stained with pinacyanole as well. Conclusion The supravital staining method with water-insoluble dyestuff is rarely found in iiterature and its staining mechanism is seldom discussed. We have dealt previously with the mechanism of Demel's supravital staining, and now have studied the nature of the supravital method with pinacyanole.

II. rnp FrxED sMEAR We reported previouslye) that mitochondria in the blood cells could be stained when a wet film of a bone marrow aspirate fixed in Champy's fluid r;r,as treated with alcoholic sudan III solutions (Romeis's fluid, Kawamura-Yazaki's fluid, 'Goldmann's fluid, etc.). Though sudan III is said to be an indifferent dyestuffst and pinacyanole seems to be a color base, both are insoluble in water and easily soluble in alcohol. So, why would not mitochondria be stained by pinacyanole .as well as by sudan III ? This second chapter concerns itself with this problem. 252 K. TAKIKA\,VA trT AL.

Material and method While the smears of Yoshida sarcoma ascites, blood or bone marrow were still wet, they were fixed in Champy's fluid (the so called wet film), rinsed in water, immersed in the pinacyanole solution in absolute alcohol for several minutes, and differentiated in 60,od alcohol, washed in water, then mounted in the gum syrup of Apathy (gum arabicum, sugar and a little thymol) or in fruit sugar. Or more easily, the wet films fixed in Champy's fluid were stained by the serum saturated with pinacyanole for one or two hours, washed in water, and. mounted in the gum syrup of Apathy or in fruit sugar. Findings In the cytoplasm of Yoshida sarcoma cell, bluish stained fine filaments and. rods are visible. These are characterized by their arrangement, that is, numer- ous around the bay of the nucleus and scarce in the periphery of the cytoplasm. Also dark blue droplets are seen scattered in the cytoplasm. The nucleus is colored purplish; nucleoli and its nucleal membrane can be clearly distinguished. Discussion In comparison with the findings of the supravitai staining with Janus. green, Altmann's stain and the phase contrast microscopy, it is found by con- sidering shape and location in the cytoplasm that the stained filaments and rods must be mitochondria. And this finding is similar to those of the supravital staining vi,ith the pinacyanole and the sudan III stain on the wet film fixed in Champy's fluid. That is to say, on the wet film fixed in Champy's fluid, mitochondria can be stained distinctly with the alcoholic pinacyanole solution or the pinacyanole serum as well as with the sudan III solution. This new mitochondrial staining method with pinacyanole is excellent in its clearness and simplicity. Fat droplets in the cytoplasm are colored dark bluish and this dark color tone is due to osmification. One merit in mounting in the gum syrup of Apathy is the facility in distinguishing the mitochondria from the nucleus by their differ- ences in coloration (the mitochondria are blue and the nucleus are purple). So pinacyanole may be either a metachromasic dyestuff or an impure one in itseif. It has been pointed out by many authors that the selection of the fixative is of first importance for the staining of mitochondria. But another condition (difficulty) in this staining method on the rvet film is that the cell membrane must be fastened to the slide as soon as it is fixed with the fixative. Among the so called mitochondrial fixatives, those which contain neither osmium tetroxide nor mercurial bichloride can not fulfil this condition. The fixatives con- taining bichloride of mercury, however, are prone to shrink the cell and so are Iiable to destroy its detail. And it has been confirmed that Champy's fluid which consists of osmium tetroxide, chromic acid and bichromate of potash is most effective in mitochondrial staining on the wet smear. When one looks at the unstained wet film fixed in Champy's fluid through a microscope, mitochondria are distinguishable, and therefore the choice of the fixative determines the success of the staining. Champy's fluid is said to preserve well the protein and lipid components in the cell and to show the fine structure MITOCHONDRIAL STAINING WITH PINACYANOLE, EMPLOYING YOSHIDA ASCITES SARCOMA CELL 253 of the cell as it is alive, hence, it is thought that Altmann's staining method €xploitslo) the difference of 'oPoren weit" (Pappenheim,'o) or, Moellendorff:10) "Dichtigkeit," 1.e., density in the structure) in formed components of the cell in staining them separately, that is to say, the acid fuchsin molecules in AIt- mann's staining fluid force their way into the most dense mitochondria and. ate held so firmly that they can not be washed away by the routine differentiation method. . Our mitochondrial staining methods with sudan III and pinacyanole have revealed that the well preserved Iipid component in mitochondria can be stained by these fat-soluble dyestuffs. Because there has hitherto been no report6) of an experiment which has succeeded in staining the lipid pattern of mitochondria, we present here our new methods.

Summary When the wet smear of cell suspensions (blood, bone marrow, ascites, etc.) fixed in Champy's fluid are treated with a pinacyanole solution in absolute alcohol followed by differentiation in 6026 alcohol, or more easily, stained by the serum saturated with pinacyanole, and mounted in the gum syrup of Apathy (or in fruit sugar), mitochondria in the cell are distinctly colored. This new mitochondrial staining method is excellent in its clearness and simplicity. Thus we proved that the fatty pattern of mitochondria can be stained with pinacyanole (and sudan III), and we stress that a choice of the fixative is of the first importance for surcess in mitochondrial staining. Conclusion Water insoluble dyestuffs (especially color bases and color acids) are not widely used in cytological stainings. The stainability of the fatty component in mitochondria, which has not been formerly proved, seems confirmed by using these water insoluble dyestuffs, pinacyanole and sudan III.

Mitochondria of Yoshida ascites sarcoma cell : the wet smear fixed in Champy's ffuid, stained in serum saturated with pinacyanole. 254 K. TAKIKA\,VA trT AL.

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