hargitt: pennaria tiarella .and tubularia crocea. 163 



II. Material and Methods. 



Tubularia crocea was collected during November and June from a 

 floating dock and adjacent piles near the mouth of the Charles River 

 in Boston, and also in April and in June from the piles of the U. S. 

 Fisheries dock at Woods Hole. Pennaria tiarella was obtained during 

 July and August at Woods Hole. Colonies of the latter were collected 

 and taken to the laboratory late in the afternoon, male and female 

 colonies being kept in separate dishes. The medusae became free 

 about seven o'clock in the evening. When the eggs had been dis- 

 charged in sufficient numbers, spermatozoa were introduced by adding 

 a pipetteful of the water from the dish containing the male colonies, 

 in which the spermatozoa were very abundant and active. No trouble 

 was experienced in getting the eggs fertilized, the desired stages being 

 therefore easily obtained. In order to secure stages earlier than 

 fertilization, it was necessary to remove medusae from the colony 

 before the time of liberation. The medasae thus artificially set free 

 were at once fixed, some of them 12-15 hours before liberation, others 

 10 hours before, and still others only a short time before that event. 



For killing, the following fluids were used: Flemming's stronger 

 mixture; aqueous solution of corrosive sublimate and acetic acid; 

 Bouin's picro-formol; Zenker's fluid. In preserving Tubularia the 

 corrosive-acetic mixture was used mostly, and gave excellent results. 

 Staining after its use was brilliant. Other fixing methods were used 

 for comparison with the corrosive mixture. For Pennaria, Flemming's, 

 Zenker's, and Bouin's fluids all gave good results, though the last was 

 the most satisfactory. 



Heidenhain's iron hematoxylin, either alone, or followed by Congo 

 red, orange G, etc., produced fine staining, and was used almost exclu- 

 sively in working out the form and condition of nuclear constituents. 

 Conklin's picro-hematoxylin did good service in staining achromatic 

 structures, such as asters, centrosomes, and the like, and gave good re- 

 sults for the study of the nucleolus. Delafield's or Ehrlich's hematoxy- 

 lin followed by eosin proved most satisfactory for the study of nucleoli. 

 For purposes of comparison, several staining methods were used in 

 every case, viz: hematoxylin and cochineal, Mallory's phosphotungstic 

 acid hematoxylin, iron Brazilin, both Auerbach's and the Ehrlich- 

 Biondi mixtures of acid fuchsin and methyl green. These last two 

 stains were of some value when used for comparison with others, but I 

 did not find that they are the safe indicators of chromatin that some 



