EMBRYOLOGY. 95 



METHODS AND MATERIAL. 



The material used in this investigation was collected in the autumn 

 of 1908 at Richmond, Ind., and in 1909 and 1910 at La Fayette, Ind. 

 The eggs were killed and fixed mainly in two solutions that are practi- 

 cally the same. The first was a saturated solution of bichlorid of 

 mercury (corrosive sublimate) in 35 per cent alcohol, 95 volumes, 

 and glacial acetic acid, 5 volumes. The second was a saturated 

 solution of bichlorid of mercury in 50 per cent alcohol, 94 volumes, 

 and glacial acetic acid, 6 volumes. The fixing fluid was raised to a 

 temperature of 75° to 80° C, poured over the living specimens, and 

 allowed to act from 5 to 10 minutes, after which it was replaced by 

 the same solution, cold, for an equal length of time. The specimens 

 were then washed in 70 per cent alcohol, in which they were kept 

 until sectioned. Gibson's fluid was found to be a very good killing 

 and fixing agent also. 



For sectioning, the following method was employed: The eggs 

 were punctured with a fine needle, dehydrated, and kept 20 to 

 30 minutes in paraffin of about 54° C. melting point. They were 

 oriented in a watch glass (that had previously been smeared with 

 glycerin) with a hot needle, under a binocular microscope, the bottom 

 of the watch glass being first quickly cooled with a little cold water. 



The eggs were cut with a Minot-Zimmermann microtome in 

 sections from 8 to 13 jj. in thickness, attached to the slide with Mayer's 

 albumen fixative, and stained with Delafield's hematoxylin or by 

 Heidenhain's iron-alum-hsematoxylin method. 



Surface views of the embryo were obtained by dissection. For 

 dissections it was found that the best results were obtained by using 

 material that had been freshly fixed and washed. Grenacher's 

 alcoholic borax-carmine was used for staining in toto. 



GENERAL DESCRIPTION OF THE EGG. 



The eggs are broadly elliptical with a slight reniform tendency. 

 They are 0.70 to 0.78 mm. in length and 0.33 to 0.45 mm. broad. 



At oviposition the egg is a very pale yellow, changing in a few 

 hours, at a temperature of 50° to 70° F., to a faint greenish color. At 

 this stage there appears an almost circular area of darker green at one 

 pole of the egg; we have termed this the "ovarian yolk," a brief 

 description of which occurs in the following pages. At the end of 24 

 hours the walls of the egg about the ovarian yolk appear denser 

 and of a deeper green. The germ band is now forming and invaginat- 

 ing. During the next 24 hours this process is completed, the egg 

 becoming a darker green in the meantime. By the third day a rod- 

 shaped body can be seen near the center of the egg. This object is 

 the submerged germ band. By the end of the third day the egg 

 becomes black. 



