52 



lY. Methods used for om* experiments ; species of 

 sponges observed. 



After several trials, we founcl that the best fluid to kill and 

 to preserve the sponges is osmic acid. Our statements are thus 

 all made after preparations in osmic acid of IV^. Of each sponge 

 we took first a small piece and looked at it in the fresh state. 

 If the flagella of the choanocytes were well moving , we decla- 

 red the sponge alive and healthy. Small pieces of about 5 mm. 

 in cubic were then cut of with a sharp knife and left in osmic 

 acid for an hour in the dark. One piece was then removed from 

 osmic acid into water for at least 24 hours. The cells are then 

 easily isolated. Another piece was rapidly washed in water and 

 then transferred inside a dialisator in a small quantity of water; 

 outside was alcohol of 90 — 96 per cent. After two or three hours 

 it was brought outside the dialisator, the latter being removed. 

 Then after one or two hours in alcohol absolutus. Again after 

 one or two hours with new alcohol absolutus inside a dialisator; 

 outside pure benzol. Thus it remains for several hours. We found 

 uo harm even after 10 or 12 hours. The piece was then brought 

 outside the dialisator and finally in pure benzol. After 2 — 10 

 hours in a mixture of equal parts of benzol and hard paraffin. 

 In this it is left for a night in an open glass, so as to allow 

 the benzol to evaporate in the stove (60^ Centigrade). Then for 

 half an hour in pure paraffin, in which it was imbedded. — 

 Sections of 5 jc«. were made with the transverse knife ; the 

 ribbons stretched on tepid water, and a couple of preferred sec- 

 tions stuck on a cover glass and perfectly dried , first on the 

 stove ^) then treated with alcohol absolutus and again perfectly 

 dried. The paraffin is then allo wed to melt ; as soon as it has done 

 so the object is dropped into benzol till the paraffin is dissolved. 

 Wash then in alcohol absolutus , then in water and the slice is 

 ready to be stained. A very convenient dye is certainly methylen- 



1) Care not to let the paraffin melt! 



