THE VITAL STAINING O F WHITE BLOOD CELLS WITH CRESYLECHT VIOLET R. SPIRIDONOVITCH l Department of A n a t o m y , Cornell University Medical College, N e w Pork City This paper is a record of results obtained by staining fresh blood cells with cresylecht violet from the National 9niline and Chemical Company. Other dyes were also used, but none of them give the interesting results obtained by Cresylecht violet .2 The method used in this investigation was suggested t o me by Doctor Chambers. It consists in flooding a carefully cleaned cover-glass with an aqueous solution of the dye (usually a 1:10,000 dilution) and then allowing the coverglass to dry. Upon evaporation of the solution the dye is left as a thin, dried film spread uniformly over the surface of the cover-glass. A drop of diluted blood is placed on this filmed surface of the cover-glass, which is then inverted over a hollow-ground slide. The blood can now be studied as a hanging-drop. The edges of the cover-glass are sealed with Vaseline. The dye on the cover-glass slou7ly dissolves and is then gradually taken up by the blood cells. The hanging-drop method has the advantage of keeping the blood cells away from the surface of the cover-glass; as is well known, contact with glass causes the blood cells to spread out and hastens their death. Both human and guinea-pig blood diluted in either Tyrode’s, Locke’s, o r Ringer’s solution were used in this investigation. All the observations were made on specimens in a Traveling fellow under the Division of Medical Education o f the Rockefeller Foundation, 1923. The dyes were secured through the courtesy of the Commission on Standardization of Stains. 367 368 R. S P I R I D O N O V I T C H warm chamber, and it was found that each dye used requires a special temperature f o r its optimum reaction, e.g., the most favorable temperature for neutral red is about 32°C.; f o r Janus green and diazin green, about 25"C., and for cresyleclit violet from 20°C. to 22°C. I wish to take this opportunity of thanking Dr. Robert Chambers for the dyes which he furnished me and for his advice and criticism. THE MAIN POINTS I N THE STAINING O F THE BLOOD CELLS The progressive action of the dye on the blood cells may be conveniently divided into the two following periods : 1)The period when the cell is still living; 2) the period when the cell is dying. This second period may be divided into the following stages: a ) The dying cell and the staining of the nucleus ; b ) the decoloration of the nucleus ; c) the persistence of the stained granules; d ) the complete disintegration of the cell. The first period The duration of the first period, while the cell is still living, depends upon the dye. Neutral red, which is the least toxic of the dyes used, gives no appreciable differential staining. The Janus green used allows the cell to live for one and a half to two hours, scarcely longer. Cresylecht violet permits the cell to live for a shorter period than the two aforementioned dyes, but this stain has the advantage of giving metachromatic coloration. I n the mononuclear and polymorpli leucocytes it was difficult to differentiate between the cytoplasmic granules and the mitochondria, the only difference being that the granuIes are larger, The hyaline cytoplasm, which during the first few seconds stained diffusely, gradually loses its color as the dye becomei concentrated on the mitochondria and the granules. During this period, while the cell is alive, the nucleus remains unstained and is sharply contoured as a colorless VITAL STAINING WITH CRESPLECHT VIOLET 369 hyaline body surrounded by the stained cytoplasmic constituents. The vital staining hinders neither the movements of the granules nor the amoeboid activity of the cells. This period of the vital staining is the most instructive and lasts longer than the following period when the movements of the cell become less active. The cell, on losing its vitality, gradually assumes a spherical form. The second period starts from the moment the nucleus begins to stain. The second period a. The dying cell and the staining of the nucleus. The assumption by the cell of a spherical form and the staining of the nucleus are the main characteristics of the dying cell. The nuclear membrane stains first, and then a nuclear network comes to view and takes up the dye. If Janus green be used, the color of the nuclear network is at first violet and later changes to a dark blue. The cytoplasmic granules lose their dye at the time when the nucleus stains. The smaller cytoplasmic granules lose their color most quickly, and it is significant that those cells which possess only fine granules tend t o have the most completely stained nuclei. The middle-sized cytoplasmic granules give up less stain and the large granules of the eosinophile cells least of all and, in accordance with this, the nucleus of the eosinophile cells becomes only slightly tinged. The stained condition of the nucleus and the tinged granules of the cells in this period remind one of the ordinarily stained blood smears. This period lasts from one to two hours. b. The decoloration of the nucleus. During this period the nucleus gradually loses the stain, while the cytoplasmic granules take it up again. The decolorized nucleus differs from that of the living cell in being granular and opaque. This period lasts about two hours. A peculiar degeneration phenomenon which occasionally occurs is the following: The hyaline portion of the cytoplasm flows out of the cell without destroying the cell membrane and accumulates outside the cell, sometimes flowing T H E ANATOMICAL EECORD, VOL. 27, NO. 5 370 R. S P I R I D O N O V I TCH around it. Occasionally this hyaline material assumes the form of pseudopodia, which break away as independent droplets. This hyaline material is always colorless. c. The persistelzce of the stained grawules. A cell with a decolorizcd nucleus maintains its form and its granules keep the stain f o r several hours. The eosinophile granules remain stained for more than fifteen hours, while the other granules lose their color somewhat earlier. d . The complete disintegration of the cell. After fifteen t o twenty hours from the beginning of the vital staining the granules finally lose their stain as the cell undergoes disintegration. VITAL STAINING W I T H CRESYLECHT VIOLET Herxhcimer ( '98) introduced cresylecht violct into histological work, but, in spite of its striking metachromasia, it has been little used. Herxheimer stained epidermal cells and found that the nuclear network stains blue, while the cytoplasm takes on a light red color. Fick ('02) used this dye for staining normal and diseased skin tissues. Williams ('23) has recently applied it t o a rapid diagnosis of unfixed and formalin-fixed frozen sections of various tissues. It is preferable to have the solution of the cresylecht violet filtered, although this dye is readily soluble in water. The cover-glass with the dried dye on its surface has a light violet tint. A drop of blood diluted in the ginger's solution rapidly dissolves this dye. The best temperature f o r vitally staining blood cells with cresylecht violet is 20 to 22°C. When the observations are made with a temperature of more than 30°C., no staining 0ccu1-s.~ a. Lymphocytes. The way these are stained differs much from that of other white blood cells. They are the first to become stained and are the first also to lose their vitality. Their capacity of absorbing the dye is also greater. ' Within two o r three seconds they become uniformly colored. After a In fixed blood smears stained with crcsylecht violet, only tllr nuelcus stains violet or dark blue. The red blood cells color a light green. VITAL STAINING WITH CRESE'LECIIT VIOLET 371 two or three minutes the mitochondria become visible on the peripheral zone of the cell, which always remains light blue, while the mitochondria are a darker blue. The mitochondria are not so easily recognized with cresylecht violet as they are with Janus green. The amoeboid movements of the cell and the brownian movements of the mitochondria are very slight. Ten to fifteen minutes later the lymphocytes become moribund, upon which their nuclei and cell membranes become dark blue. The mitochondria are not discernible in a dead cell. At this time all other white blood cells are only beginning to show vital staining. The decoloration and disintegration is also more rapid in the lymphocytes than in the other white blood cells. b. Large monotauclear Zeucocytes. These cells stain more quickly than do the polymorph and the eosinophile leucocytes. Their period of vital staining lasts about thirty minutes. The iiumerous granules and the mitochondria become violet. After thirty minutes the nucleus begins to take the dye, while the cytoplasmic granules lose the stain. The decoloration of the nucleus is accompanied by the restaining of the granules. The decolorized nucleus is opaque, and the granules keep the dye f o r several hours until the cell disintegrates. c. Neutrophile polymorph leucocyies. These stain in the same way as the mononuclear cells, but the vital and supravital staining periods are somewhat longer. During the vital staining these cells and their granules are less active in their movements than are the mononuclear cells. The granules of these cells are of various sizes. The mitochondria and the smaller granules are violet, while the large granules are bluish-violet. The large granules vary in number in different cells and, when they are numerous, give the cell a bluish tinge. I n the neutrophile cells of guinea-pigs the amphophilic granules are easily seen with cresylecht violet, for they assume a reddish-violet tint and resemble the granules of the eosinophile cells (cf. next subheading). They differ from the eosinophile granules in losing their stain simultaneously with the neutrophile granules. 372 E. S P I R I D O N O V I TCH After a period of thirty t o sixty minutes, the nucleus of a neutrophile cell begins to stain, but it never stains as deeply as that of a mononuclear cell. The cytoplasmic granules, and especially the larger ones, also do not lose their dye t o the same degree as the granules of the mononuclear cells. The nucleus becomes dark violet and keeps the dye €or two or three hours, after which it loses its stain, while the granules remain stained f o r several hours more. The disintegration of the cell occurs after fourteen to sixteen hours. d . Eosinophile cells. The granules of these cells are brought out strikingly with cresylecht violet. I n man they are red and in the guinea-pig they are reddish-violet. The vitally stained eosinophile cells exhibit amoeboid movements, the granules following the movements of the cytoplasm. The period of the vital staining lasts much longer in the eosinophile cells than in the other blood cells. After one hour the nucleus begins to color a light violet, but it is never sharply delineated and its chromatic structure remains obscure. This is in marked contrast to the mononuclear and neutrophile cells. The faintly stained nucleus gradually becomes decolorized after two or three hours. The granules remain stained for about twenty hours, and only lose their red tint when the cell finally disintegrates. e . Basophile cells (nzast cells). The granules of these cells are dark blue. The movements of the cells and their granules are similar to those of the eosinophile cells. The period of the vital staining is somewhat shorter than that of the eosinophile cells. The nucleus stains after thirty to forty minutes, but is stained more lightly than in any of the other cells, and the basophile granules do not lose their blue dye. The decoloration of the nucleus and the disintegration of the cell occurs earlier than in the eosinophile cells, f. Red blood cells. Under favorable conditions, these do not change in shape and they absorb no dye for the first two hours. After that they begin to swell and, if the preparation be allowed t o dry, they assume a varying pinkish-blue tint. VITAL S T A I N I N G WITH C R E S Y L E C H T VIOLET 373 g. Platelets. At first these are difficult to find, but the dark blue staining of their membranes makes them discernible after ten to fifteen minutes. The stained platelets move about and easily change their form. They sometimes contain several mitochondria which, apparently, possess independent movements. The mitochondria are blue and are more easily seen when stained with cresylecht violet than with Janus green. They keep the stain for a long time, and during the decoloration period the surface membrane of the platelets remains stained longer than the mitochondria. CONCLUSIONS 1. A special method for cover-glass staining with vital dye is described. 2. Cresylecht violet is a metachromatic dye and stains the living white blood cells in three colors: blue, violet, and red. The granules of the monuclear and polymorph cells are violet, those of the eosinophile cells are red, and those of the basophile cells are blue. These results were obtained when the cells are kept at a temperature of 20 to 22°C. 3. The nucleus stains only when the cell dies. With cresylecht violet it stains violet, except in the lymphocytes where it has a bluish tint. I n the dying cell, as the stain enters the nucleus, the smaller cytoplasmic granules lose some of their stain. Later, when the nucleus decolorizes, the small granules again become stained. The larger granules maintain their color throughout. Upon disintegration all color is lost. LITERATURE CITED FICK,T. 1902 Ubcr metneliromatische Farbung der Keratohyalins durch Kresyleehtviolett. Centralbl. f. Allgem. Pxthol. und pathol. Anat., Bd. 13. HERXHEIMER, K. 1899 Ueber die Structur des Protoplasmas der merisehliclien Epidermiszelle. Archiv. f. mikr. Annt. und Entw.-gesch., Bd. 53. WILLIAMS,B. G. R. 1923 Cresylecht violet, a rare dye. Jour. Lab. Clin. Med., vol. 8.