6 BULLETIN OF THE BUREAU OF FISHERIES. 



Fusion of the granular cells begins immediately and in a few minutes' time most 

 of them have united to form small conglomerate masses which at the surface display 

 both blunt and elongated pseudopodia (fig. 22, pi. iv). These masses soon begin to 

 incorporate the neighboring collar and hyaUne cells. One sees collar cells sticking 

 fast by the end of the long flagellum to the conglomerate mass (fig. 22). Other collar 

 cells are attached to the mass by short fiagella. Still again only the body of the collar 

 cell projects from the mass while there is no sign of the flagellum (fig. 24). Similarly 

 spheroidal hyaline cells of many sizes fuse with the granular conglomerates. 



The small conglomerate masses first formed early begin to fuse with one another, 

 while they still continue to incorporate outlying free cells. The space under the cover 

 glass thus soon becomes occupied with numerous small balls or masses (fig. 24, pi. iv), 

 which are of a syncytial nature. As the sequel shows, these masses continue to unite 

 and eventually restore or regenerate the sponge. They may be spoken of therefore as 

 masses of regenerative tissue, and the observations already described make it plain 

 that they are composed chiefly of the spheroidal granular cells or amoebocytes, but that 

 other cells, collar cells in particular, enter into their composition.'^ 



The small syncytial masses of regenerative tissue produced in the way described 

 attach with some firmness to the substratum. In order to watch their further history 

 they must be kept healthy, and with this point in view it is advisable to proceed in 

 the following fashion from the beginning. After the cells squeezed out from the sponge 

 have settled over the bottom of the dish, the water is poured off and fresh sea water 

 added. This should be done shortly, 10 to 15 minutes, after the cells have been squeezed 

 out. By this time the fusion of cells has progressed so far that the tissue exists in the 

 shape of innumerable small conglomerate masses with free cells between. The tissue 

 is easily handled. It may be sucked up with a pipette and then strewn over cover 

 glasses, slides, cloth, watch glasses, shells, etc. 



For the purposes of observation it is best to strew the tissue sparsely over slides 

 and covers. But if one w'ishes really to breed sponges, it is better to strew the tissue 

 more thickly over slides or clean oyster shells. The slides, covers, whatever is to be 

 used, are placed in a large dish filled to about the depth of 2 inches with clean sea water. 

 The tissue is dropped from the pipette. It sinks down through the water on to the 

 slides, to which it at once begins to attach. Attachment is at first easily broken and 

 for about half an hour all disturbance of the water must be avoided. At the expiration 

 of that time the slides should be gently removed from the water and held for a moment 

 in such a position that they drain. The draining off of the water causes the tissue to 

 sink closer to the substratum, to which it makes a firmer attachment. The object with 

 its coating of sponge tissue is now gently replaced in a dish of fresh sea water, where it 

 should lie for about 24 hours. During this period the water should be changed several 

 times, or the object may be kept in a running aquarium, in which it should be pro- 

 tected from any considerable agitation of the water. After a day the attachment of the 



" For a brief discussion of the question as to the fundamental nature of this regenerative tissue see my paper: On some 

 phenomena of coalescence and regeneration in sponges. Journal of Experimental Zoology, vol. v, 1907, no. 2, p. 250-252. 



