40 Papers from the Marine Biological Laboratory at Tortugas. 
ponoé esculenta, a sea-urchin very common in the shallow waters of the reefs 
in the vicinity of Dry Tortugas, Florida. I am under obligations to Dr. 
Alfred G. Mayer, Director of the Laboratory at the above place, for daily 
favors and many helpful suggestions. 
METHODS. 
As is now well known, when the immature ova of star-fish are shaken 
out of the ovary into sea-water they immediately begin to form the polar 
bodies. In ovaries opened during July and August I found from 50 to 70 
per cent of the odcytes at the stage of development where they could be 
thus induced to mature. The eggs were placed in water which was kept 
by frequent changes as nearly as possible within the limits of normal ocean 
temperature. In every case the development was allowed to proceed to the 
early segmentation stages, so as to assure a large percentage (about 60 per 
cent) of normal stages among the eggs previously preserved. In some 
cases sperm was added to the sea-water, but produced no noticeable effect, 
beyond the formation of a fertilization membrane, until after the second 
polar body was formed. 
Ovarian material was fixed in Gilson’s fluid, sublimate acetic, picro-acetic, 
and picro-aceto-sulphuric mixtures, and Bouin’s solution. The sublimate 
acetic and picro-aceto-sulphuric mixtures proved most serviceable in that 
the coagulation product was less coarse and the relations of nucleolus, 
nucleus, and cytoplasm continued intimate and their internal structure un- 
disturbed. 
The stains employed were Heidenhain’s iron hematoxylin followed by 
orange G, Delafield’s hematoxylin with Congo-red counter-stain, borax car- 
mine with orange G counter-stain, Auerbach’s stain, and Flemming’s triple 
stain. Heidenhain’s iron hematoxylin with orange G yielded by far the 
most satisfactory results. Auerbach’s stain was very serviceable in that it 
differentiated beautifully between definite chromatin and plastin. Flem- 
ming’s triple stain proved very unreliable and generally unsatisfactory on 
account of its varying reaction. Still other combinations of stains were 
employed, as will be noted in subsequent descriptions. 
The maturing eggs were fixed at intervals of 10 minutes through a 
period of 2 hours. Several series were put up at intervals of 5 minutes 
through a period of 1 hour; and still others at intervals of 30 minutes 
through a period of 3 hours. The results from four complete and several 
partial series are wholly in accord with each other. Besides the above- 
mentioned fixing reagents used for the ovarian material, Flemming’s strong 
solution was also employed. In the case of the free ova, also, the sub- 
limate acetic and picro-aceto-sulphuric mixtures proved most satisfactory 
in that they caused less disturbances within the cell substances. Flemming’s 
solution, as also Gilson’s fluid, preserved beautifully the mitotic figures, but 
eS ee 
