32 W. WOODLAND. 



living Amphiuras being obtained, the disc of eacb animal 

 was cracked, so as to permit the free access of the fixing, 

 staining, and preserving reagents to the young animals 

 contained within the bursas, and these were then fixed with 

 1 per cent, osmic acid, stained with picro-carmine, and pi'e- 

 served in 90 per cent, alcohol in the manner already described 

 in previous studies. Some of my best slides, however, have 

 been obtained by merely fixing the live Amphiuras in absolute 

 alcohol, subsequently staining for a fortnight or more in a 

 saturated solution of safraniu in absolute alcohol and Avashing 

 out in absolute alcohol for a month or so after (if the alcohol 

 be warmed less time will suffice). In many cases I also 

 employed lichtgriin as a plasma stain, but, if employed, the 

 solution in absolute alcohol must either be very weak, or, if 

 a saturated solution, the Amphiuras must only be immersed 

 in it for a minute or so, otherwise the tissues become opaque. 

 Lichtgriin, when successfully employed, undoubtedly gives 

 the best results ; it is, however, quite possible to Avork without 

 it, though in this case it is more difficult to be quite certain 

 on occasion as to whether a particular cell belongs to a 

 particular spicule or not. When the Amphiuras have been 

 stained the discs are opened and the young ones extracted ; 

 these are then transferred to xylol, and finally mounted in 

 balsam. In general only the youngest Amphiuras (about 0*5 

 mm. in diameter; see fig. 1) yield satisfactory results, though 

 now and again it is possible to observe young spicules in the 

 arms of the older Amphiuras. It is surprising how very few 

 really satisfactory young Amphiuras are obtainable from 

 numerous parents : from at least five or six hundred adults 

 I have only managed to secure about a score of young ones 

 showing the origin of spicules in an unmistakable manner. 



The above-described methods of preparation were also 

 employed in the examination of the specimens of Ophiothrix 

 and Echinus. 



My Antedon material consisted of very young specimens 

 which, after having their discs opened, were prepared by the 

 osmic and picro-carmine method. The imperforate thin plate 



