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Iron-Hematoxylin Stain 11-Day Embryo Flemming Fixation

plate, germ-cell, fig, granulocyte and mesenchyma

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In view of what was said above it seems clear that the best way of approach to the problem was through the 11-day embryo preserved in Flemming's fluid and stained with iron-hematoxylin. The next step involved a comparative study of embryos of this same stage fixed with Helly's fluid and stained respectively with the iron-hematoxylin and the Giemsa mixtures. Thus the criteria for identifying the primordial germ-cell were established and the previous and later history could then be traced with comparative ease.

This stage corresponds closely with von Berenberg-Gosaler's illus tration (fig. 9) of a lizard embryo. Primordial germ-cells are present among the entoderm-cells of the closed hind-gut and in the surrounding mesenchyma; in the mesentery, both in the mesenchyma and its mesothelial covering; and in the primitive genital gland, both among the peritoneal cells and in the subjacent mesenchyma (see fig. 2, plate 6). Figure 1, plate 1, is a composite drawing including the several types of cells with which we are now concerned, all from the same immediate region of the mesentery. We may compare first cells a and b, a typical germ-cell and a typical eosinophil granulocyte, both in the mesenchyma of the mesentery. Cell-type b may appear at any stage after the second day of incubation in any part of the yolk-sac or embryonal mesenchyma, including the blood-vessels. At first sight it would seem to correspond to what Swift described as a primordial germ-cell circulating in the blood-vessels of the young chick embryo. It contains an attraction sphere (i), a spherical deep-staining chro matic nucleus, and abundant small spherical granules (apparently comparable to the granular mitochondria of germ-cells) ; but the re semblance is only very superficial and no confusion need arise.

Cell a is a typical primordial germ-cell. Ordinarily, in the inactive condition, it has an oval form, rather than the more nearly spherical form of the granulocyte in the same condition. Moreover, it is nearly twice the size of the largest granulocyte. Its nucleus is vesicu

lar, contains a very characteristic finely granular karyoplasm, and stains only very lightly in basic dyes. The radial disposition of the rows of oxyphilic chromatin granules is striking. There is present usually only one large, very chromatic, spherical or oval nucleus; and one (or sometimes several) much smaller chromatic accessory nucleoli; and usually one (sometimes several) pale plastin nucleoli. The cytoplasm is more or less crowded with larger and smaller yolk spherules, staining more or less deeply. It contains also abundant granular, bacillary, beaded, and filiform mitochondria (fig. 2a, plate 1).

The granules of the blood granulocyte, on the contrary, are quite uniform in size and in their deep-staining quality. The nucleus of the granulocyte also is relatively smaller, contains a relatively coarse chromatic net-work, and stains much more deeply as a whole in basic dyes. In anticipation of what follows it may here be stated to advan tage that primordial germ-cells of earlier stages, including the 2-day stage, are of approximately the size of those of the 11-day stage, only occasionally slightly smaller. The germ-cells are therefore always larger than the largest granulocyte (plate 1, figs. lb and 2b), the largest hemoblast (plate 1, fig. 1h), and the largest erythroblast (plate 1, fig. 5). The nucleus of the germ-cell also is quite different from that of any of the cells in close proximity to which it may be found—granu locyte (b), mesothelium (M), mesenchyma, hemoblast (h), erythro blast (fig. 5), and erythrocyte (plate 1, fig. le). It resembles closely only the young entodermal cell, especially in respect of its nucleus, from which cell the germ-cell can, however, be distinguished by criteria which will be given below. Moreover, the granulocyte may assume most varied shapes, at different stages of amceboid progression; the germ-cell, while also capable of amceboid movement, becomes less irregularly modified, extreme forms being represented in figure 3, aa, ab, ac, plate 1.

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