40 Papers from the Marine Biological Laboratory at Tortugas. 



ponoc esculcnta, 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 oocytes 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 Benin'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 varving reaction. Still other combinations of stains were 



* *j 



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 i 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 



