THE BLOOD OF ALLIGATOR MISSISSIPPIENSIS ALBERT M. REESE Department of Zoology, West Virginia University EIGHT FIGURES METHODS In this study of the blood of the alligator both fresh and stained preparations were used. The blood was obtained from an animal kept in the aboratory, by making a small slit between the ventral abdominal scales; the wound quickly healed. This operation was repeated whenever a new preparation was needed. By sealing the cover with a ring of oil, to prevent access of air, the fresh blood could be kept in normal condition for many days, during which period the amoeboid activity of the leucocytes could be studied. In making stained preparations various fixing fluids were used, but the ordinary dried smears gave the best results. Of the stains used the best results were obtained with hematoxylin and eosin and with Wright’s stain. THE ERYTHROCYTES The red cells of the alligator’s blood are, as is well known, of the usual elliptical form seen in the lower vertebrates. According to Gulliver (2) there is some variation in the cells of closely related species. He found (3), as will also be noted below, that the corpuscles of dried blood are appreciably smaller than those of fresh blood. He says that in Crocodilus acutus and in an unknown species from Vera Cruz the length of the corpuscle is somewhat less than twice the breadth. 37 T H E ANATOMICAL RECORD, VOL. 13, NO. 1 38 ALBERT M. REESE On the other hand, Mandl (4) who studied C. lucius says the length is two or three times the width. In only three cases of several dozen measurements made of the corpuscles of A. mississippiensis did the writer find the length as much as twice the width, and in no case did it approach three times the width. In C. acutus Gulliver gives the average length of the corpuscles as 20.31+ micra; the average width as 10.93f micra. In C. fissipes the average length was 19.85+ micra; the average width was 10.79 micra. As will be seen below, these variations arenot nearly so great as those seen among corpuscles from the same individual animal in the Florida Alligator, A. mississippiensis. Milne-Edwards (5) gives the following measurements, presumably averages; A. sclerops, length 23.80. micra; width 13.33 micra; A. lucius length 20.88 micra; width 11.11 micra. In A. mississippiensis a considerable number of measurements was made, both of fresh and of stained corpuscles. The thickness of the corpuscles was also measured in a number of cases; this can be done only with the fresh blood where corpuscles can occasionally be seen in profile. The average length of the fresh corpuscles was found to be 20.77 micra the average width 12.78 micra; and the average thickness 4.17 micra. As noted above, and as will be seen later, these measurements are apparently greater than those of stained corpuscles. The longest corpuscle found in the fresh blood was 24 micra; the widest was 14.00 micra; the thickest was 5 micra; the shortest was 18.50 micra; the narrowest was 11micra; and the thinnest was 3.60 micra. Among the stained corpuscles the average length was 18.69f micra; the average width was 10.85 micra, both measurements being noticeably less than in the fresh blood. The longest stained corpuscle measured wa,s 22.10 micra; the shortest stained corpuscle was 14.80 micra in length, only 0.8 micra more than the width of the widest fresh corpuscle. The widest stained corpuscle was 12.80 micra; the narrowest was 8.10 micra. + BLOOD OF ALLIGATOR MISSISSIPPIENSIS 39 Among all the corpuscles, both fresh and stained, the one that showed the greatest difference between the length and the width was one that was 22.10 micra long and 10.40 micra wide. On the stained slides, for obvious reasons, it was not possible to measure the thickness of the corpuscles, but the length and width of the nucleus were measured in each case. The greatest variation in the length of the nuclei was from 3.80 micra to 6 micra; in width from 2.80 micra to 4.30 micra; the average length was 4.85 micra, the average width was 3.66 micra. The appearance of the erythrocytes, as seen in the flat, is shown in figures 1, l a , 1 b, and Id-h, drawn, as were all of the figures, with a camera lucida under an oil immersion objective from a stained slide. The profile view, figure 1c, WAS, of course, drawn from a slide of fresh blood. As would be expected from the measurements given above, the ellipse varies considerably in different corpuscles. In the profile the central thickening is plain but the nucleus could not be determined. The cytoplasm, so far as could be determined, was homogeneous, though it was examined under a magnification of 2300 diameters. The cytoplasm was so transparent that, on the ordinary stained slide, when one corpuscle lay over another the nucleus of the under corpuscle showed with the same apparent distinctness as that of the upper cell. This structureless condition is in contrast to that described by Bryce (1) in Lepidosiren, where he figures a clear band beneath the membrane, a fine network throughout the cytoplasm, and one or more clear areas and vacuoles in the cytoplasm. Whether examined under a magnification of 1000 or of 2300 diameters, the cytoplasm in A. mississippiensis appeared the same. Possibly more refined methods of technic might have brought out some details of structure in the cytoplasm. The nuclei, as indicated by the measurements and as seenin figures 1, 1a, 1b, etc., vary both in size and shape, though they are usually ellipsoidal. They stain easily and darkly but not homogeneously; sometimes small unstained or more lightly stained areas are scattered fairly evenly throughout the nucleus, as in figure 1 ; sometimes larger and more irregular areas are 40 ALBERT M . REESE seen in various parts of the nucleus as in figure l a . Occasionally a nucleus is located at one end of the cell instead of its usual central position, and sometimes a nucleus is seen at each end of the cell, figure -1 g. No instance of mitosis was seen on any slide examined, but erythrocytes are occasionally found with two closely adjacent nuclei, figure 1 1 9 , which would seem to have just resulted from an amitotic division. Figure I f seeins an evident case of amitotic division of the nucleus just before the completion of the process. While the cells in which two nuclei are found are usually of large size and elongated form, but one case could be found in which there was indication of a division of the cell body. This cell is shown in figure 1 la; it is possibly an artifact, but it is difficult to see how the nucleus could have been pulled apart by artificial means as is shown in the figure. Quite infrequently erythrocytes of the form shown in figure 1 d are seen. I t would, at first, seem possible that these were halves of just-divided cells, but if this were the case they should frequently be founds in pairs, while, as a matter of fact, it is but seldom that, two of them are found in the same microscopic field. It is possible that t6ey may be comparable to the spindle cells found in frog’s blood, but it seems more likely that they are merely artifacts. THE LEUCOCYTES On a slide of fresh blood, mounted with a ring of oil to prevent access of air, aa noted above, the amoeboid activities of the same white corpuscle may be studied for several days, though after a few days the motion is so slow that it can only be determined by making a series of drawings at intervals of several minutes, as must sometimes be done to demonstrate the changes in shape in amoeba. In the fresh blood it is difficult to identify the various types of leucocytes that may be seen in the stained blood, not only because of the lack of stain but also because the pseudopodia are usually more or less withdrawn in the stained blood. Figure 2 shows one type of leucocyte as seen in fresh blood. The general outline of the cell is circular and a number of small, BLOOD OF ALLIGATOR MISSISSIPPIENSIS 41 sharply-pointed pseudopodia project from its periphery. Being unstained, the nucleus is indistinct or invisible, but one or more small vacuoles may be seen. The cell i s filled with fine, unevenly distributed granules which change their appearance as the cell changes its shape. Such a cell, while it changes its shape but little, changes quite rapidly-about as fast as the changes seen in an active leucocyte in frog’s blood. Another type of leucocyte, seen in fresh blood, is shown in figure 3; it is coarsely granular and changes its shape quite rapidly and markedly. In stained preparations of alligator’s blood several types of leucocytes may be distinguished. Of these the most numerous is shown in figure 4;since it stains with hematoxylin rather than with eosin it might be thought to be an extruded nucleus from an erythrocyte except that it is several times the bulk of such a nucleus. It is of fairly large size and is usually circular in outline though the shape is variable. It is possible that it is a corpuscle in which the nucleus is very large and the cytoplasm is so reduced as to be invisible. . This would seem possible from the fact that occasional cells are found with a very large nucleus and a very thin peripheral zone of protoplasm. Of almost, if not quite, as frequent occurrence as the cell just described is a smaller type shown in figures 5, 5 a, 5 b, and 5 c. This cell may, perhaps, correspond to the lymphocyte in the human blood. It varies considerably in size and shape but contains an oval or circular nucleus and a small amount of cytoplasm which generally gives the cell a pointed or spindle form, as seen in figures 5 b and 5 G. Another type of leucocyte that is fairly common is shown in figures 6 and 6 a. These forms might, perhaps, be called mononuclear leucocytes; they are large, some of them being larger than any of the other types. The cytoplasm is clear or very finely granular, and stains, with eosin, a pale pink color. The nucleus is very large and of an oval or circular outline; it does not stain so darkly as the nucleus of the erythrocyte. The outline of the cell is usually circular or polygonal. The most striking in appearance of all the leucocytes, and, possibly with one exception, the least numerous, is the type 42 ALBERT M. REESE that may be compared to the eosinophile cell of mammals. It is a large, usually circular cell that may at once be recognized by its coarsely-granular cytoplasm that take a strong pink color with eosin. The nucleus is usually round or oval, and generally lies close to one side of the cell, as shown in figure 7. Occasionally two nuclei in a single cell may be seen, figure 7 b, as though by division of the larger nucleus; and an occasional elongated nucleus, as seen in figure 7 a,would seem to indicate an impending amitotic division. No case was seen in which there was any indication of the division of the cell as a whole. In many of these eosinophille cells, especially in those in which the cytoplasm did not take the stain, there was seen a heavy outline, like a thick cell wall, possibly caused by a peripheral zone of denser protoplasm; this appearance was usually most marked on the side of the cell farthest from the nucleus. This type is the most uniform in size and shape of any of the leucocytes. The least numerous type of leucocyte, if indeed it be a distinct type, is shown in figures 8, 8 a, and 8 b. Among the tens of thousands of erythrocytes seen, pn several different preparations, but three of this possible type of leucocyte were seen; it is this extreme rarity that raises the doubt as to their being a normal type of corpuscle. They are all of rather small size and not very irregular outline. The cytoplasm is clear or very finely granular. The nucleus, or nuclei-figure 8 b shows no less than eightare so dark as to be almost a solid black. Whether these are really a normal element of the blood or are some artifact or other abnormality it is difficult to determine. LITERATURE C I T E D (1) BEYCE,T. H. 1904-5 The histology of the blood of the larva of Lepidosiren petradoxa. Trans. Roy. Soc. Edinb., vol. 41, no. 9, pp. 291-311 and 435-69. (2) GULLIVER,G. 1840 On the blood corpuscles of t h e Crocodilia. Pro. Zool. SOC.London, vol. 8, p. 131. 1842 On the blood corpuscles of the British Ophidians, Reptiles and (31 other oviparous vertebrates. ibid, vol. 10, pp. 10s-11. (4) MANDL, 1839 Note sur les globules sanguine d u Prothe e t des Crocodiliens. Ann. des Sc. Nat., 2 Serie, T. 12, p. 289. (5) MILNE-EDWAIRDS, A. 1856 Note sur les dimensions de globules du sang chez quelque vertebres. Ann. des Sc. Nat, T. 5, pp. 165-7. - BLOOD OF ALLIGATOR MISSISSIPPIENSIS 2 3 8a 43 4 6a EXPLANATION O F FIGURES All of the figures were drawn with a camera lucida under the same magnification, i$ oil immersion objective and no. 8 compensating ocular. Figs. 1 , 1 a, 1 b Three views of erythrocytes seen i n the flat; figure 1c is a normal erythrocyte seen i n profile, drawn from a slide of unstained and living blood. Figure 1 d is one of the rhther uncommon pointed erythrocytes, which may be 44 ALBERT M. REESE siniply a n artifact. 'Figure 1 e is a red cell with, apparently, a just-dividcd nucleus. Figure 1 f is a cell in which the nucleus is i n process of division. Figure 1 g is a red cell with a. nucleus a t each end. Figure 1 h represents the single case that, was found that. seemed t o show a n erythrocyte in which the entire cell was in process of division. Figs. 2 and 3 Rcpr'esent two types of leucocytes drawn from living blood while cxhil)iting amoeboid motions; figure 3 represents a more active cell t h a n figurc 2, and one in which t,he granules are coarser. Fig. 4 Represents a type of doubtful character, which may be simply a cell with a n enormous nucleus and almost no cytoplasm; i t is the most common of the leucocytes. Figs. 5> 5 a, 5 b , and 5 c Represent a type of small leucocytes very abundantly represented. Figs. 6 and 6 a Ruepresent a type of large mononuclear cells t h a t may possibly be thc same as the one shown in figure 4. Figs. 7 , 7 a,and 7 h Show three very characteristic cells t h a t seem t,o correspond t o the eosinophile cells of mammalian blood; they are very coarsely granular and generally stain strongly with Eosin. Figs. 13, 8 a, and 8 h Represcnt a type of small and unusual cells t h a t are so seldom seen a s t o make it seem doubtful t h a t they are a normal constituent of blood.