446 WILSON. [VoL. III. 
placed in 70 per cent alcohol for a day, and kept permanently in go per cent 
alcohol. 
For permanent staining no method has proved so satisfactory as borax- 
carmine followed by hematoxylin. After being deeply stained in the carmine 
(12 hours), and extracted in acid alcohol in the usual manner, the embryos 
were treated with extremely dilute ammoniacal alcohol for a few minutes, to 
neutralize the free acid, and were then stained in very dilute Kleinenberg’s 
hematoxylin (12 hours or more). In case of overstaining with hematoxylin, 
the color may be again extracted with acid alcohol, after which the specimens 
are again treated with ammoniacal alcohol. This process, following treatment 
with Perenyi’s fluid, gives beautifully clear preparations, which are specially 
favorable on account of the clearness with which the cell-outlines are shown. 
It has been found desirable to embed the specimens for sectioning, as soon as 
possible after hardening, and to reduce the time of immersion in melted paraf- 
fin to a minimum (Z.e., not more than Io or 15 minutes). 
For surface-views of the germ-bands, the borax-carmine stain should be 
very deep, and the hematoxylin very slight, so as to give the specimen only 
a purplish color, not a dark blue. The germ-bands are dissected off on the 
slide, in strong glycerine. This method has, in my experience, given far better 
results than that of osmic acid followed by Merkel’s fluid, so successfully used 
by Whitman in the study of Clepszne. 
For the study of entire specimens of the young stages, I have found Pe- 
renyi’s fluid followed by alcohol, water, very dilute iodine solution, and gly- 
cerine, to give results superior beyond comparison to those attained by any 
other method. The iodine colors the protoplasm pale yellowish brown, the 
cell-outlines are clearly marked, and the nuclei are stained deep brown. In 
time, most of the iodine is precipitated in the form of deep brown spheres, 
which mar the clearness of the preparations, but such specimens may be after 
wards stained with carmine, etc., sectioned and mounted in balsam in the 
usual manner, and give perfect satisfaction, even after a stay of two years or 
more in the glycerine. 
LIST OF PAPERS REFERRED TO. 
1. BALFouR, F. M. Comparative Embryology, I., II., 1880, 188r. 
2. BaTeson, W. The Early Stages in the Development of Balanoglossus 
(sp. incert.). Quart. Fourn. Micr. Sci., XXIV. 1884. 
3. Id. The Later Stages in the Development of Balanoglossus Kowalevskii. 
Quart. Fourn. Micr. Sct.. XXV. Supplement, 1885. 
4. BEDDARD, F. Note on the Paired Dorsal Vessel of Certain Earthworms. 
Proc. Roy. Phys. Soc., Edinburgh, VIII. 1885. 
5. BEeRGH,R.S. Die Metamorphose von Aulastoma gulo. Aré. a. d. Zod) 
Inst. Wiirzb., VII., 3. 1885. 
6. Id. Die Exkretionsorgane der Wiirmer. Kosmos, 1885, II., 2. 
7- Id. Die Entwickelungsgeschichte der Anneliden, etc. Kosmos, 1886, 
EI. 6; 
