318 Papers from the Department of Marine Biology. 



Two chief reasons led to the selection of the loggerhead-turtle embryo 

 for this investigation: (1) the possibiUty of obtaining a large and 

 closely graded series of stages; the number of eggs per nest is near 100; 

 development is comparatively slow, the incubation period being about 

 8 weeks; (2) I had already familiarized myself in previous works with 

 the embryonic blood-cells of certain Chelonia (1913) and also with the 

 male germ-cells in a study of the spermatogenesis of several species 

 of turtles (1914). 



The object in using Helly's fluid as a fixative almost exclusively was 

 to preserve the cytoplasmic granules, both albuminoid and lipoid, 

 that is, blood-cell granules and mitochondria, for subsequent differen- 

 tial staining with the Giemsa stain and with iron hematoxyhn. Paral- 

 lel series were prepared with these two stains, with the expectation 

 of facihtating the differentiation between hemoblasts and gameto- 

 blasts. This technic was very serviceable, but did not yield quite the 

 fine uniform results hoped for. As concerns the hemoblasts and granu- 

 lar blood-cells (eosinophiles) nothing better could have been desired; 

 but the cytoplasmic contents of the primordial germ-cells were only 

 indift^erently preserved. The fixation period should probably have 

 been prolonged for 3 or 4 days beyond the usual 24 hours, especially in 

 the case of the older embryos. Only very rarely were mitochondria 

 and yolk-globules preserved in the later stages. Especially in the 

 germ-cells were the yolk granules dissolved, leaving thus a greatly 

 vacuolated and distorted cytoplasm. But the Helly's fluid and the 

 subsequent treatment involved in the paraffin technic had a very 

 variable effect upon the yolk-granules at different stages. The same 

 type of cell might contain many or only a few, or no yolk-granules. 

 In the latter case the yolk-content had been entirely dissolved. Germ- 

 cells of the 2-day stage of incubation showed many yolk-spherules; 

 by the fourth day no germ-cells contained yolk after this fixation. 

 Obviously the later steps in yolk metabolism were more susceptible 

 after Helly's fixation to the solvent action of the alcohols and oils 

 used in the paraffin technic. The young germ-cells evidently contain 

 less readily soluble yolk than other cells, with the exception of the 

 entodermal cell of the area opaca. In tissues fixed with Helly's 

 fluid and stained with iron-hematoxylin the germ-cells in early stages 

 therefore stand out clearly from among the entoderm-cells of the area 

 pellucida, and accordingly this technic proved to be very favorable for 

 the study of this early, most important, stage. 



Some embryos of the eleventh and the twenty-fifth day of incubation 

 were fortunately preserved in Flemming's fluid. It so happens that 

 the 11-day stage of the incubation period is crucial from the viewpoint 

 of the greatest abundance of primordial germ-cells in the area including 

 the closed hind-gut, the mesentery, and the primitive gonad. In these 

 cells mitochondria as well as yolk-granules are well preserved; and so 



