xvi ECHINODEKMATA 459 



staining agents to act on tissues so impregnated. It is found in 

 practice that if the sections be immersed in a strong solution of 

 borax-carmine for twenty-four hours they absorb little or no stain ; if, 

 however, after this treatment they are transferred to a dilute-solution 

 of Grenadier's haeinatoxylin in distilled water they will become 

 densely stained. 



The advantages of this method are, (1) that the unsaturated chromic 

 acid in the bichromate of potash acts on calcareous deposits so slowly 

 and gently that they are dissolved without any accumulation of 

 carbonic acid gas resulting, and without any tearing of the surround- 

 ing tissues, because such carbonic acid as is produced is dissolved in 

 the water of the solution as fast as it is produced ; (2) that the form of 

 the cells and tissues is preserved with the most exquisite faithfulness, 

 such delicate structures as fine flagella being clearly visible when an 

 immersion lens is used. 



If a section of a larva preserved in osmium tetroxide and Miiller's 

 fluid be compared with a section of one preserved in corrosive sublimate 

 it will be seen that in the latter there is what one might almost term a 

 clotted appearance, i.e. the delicate cells of the connective tissue 

 tend to cohere in lumps, and the cytoplasm is seen, when subjected 

 to minute examination, to be somewhat shrunken, and to stain badly ; 

 whereas in the osmium tetroxide preparation the individual forms of 

 these connective tissue cells are exquisitely preserved, and their 

 cytoplasm is deeply stained and sharply distinguished from the 

 semi-fluid ground substance in which they are immersed. 



The disadvantages of the method are, (1) that it renders the objects 

 so treated very brittle, and hence arises the absolute necessity of 

 embedding in celloidin. For this reason it is less suitable for dealing 

 with yolky eggs, like those of Cribrella and Solaster, than with 

 comparative yolkless eggs, like those of Asterias, since the combina- 

 tion of the osmium tetroxide with the particles of yolk is extremely 

 hard and resisting to the knife ; (2) if applied to embryos of any size 

 the osmium tetroxide is apt to form a hard black crust on the 

 outside, and to prevent the penetration of the fixing fluid to the 

 interior of the specimen, which is thus badly preserved. For this 

 reason we have sometimes used a dilute solution of osmium tetroxide 

 (25 per cent), and then allowed it to act for a considerable time, 

 because the fixative is thus enabled to penetrate to the interior. 



For the preservation of the delicate pelagic larvae which occur 

 in the life-histories of the least modified Echinoderms no other 

 'method gives results of equal value to this one. The difficulty 

 of brittleness can be got over by the use of the celloidin-paraffin 

 method of embedding, by good razors, and by restricting the period 

 of embedding in the hot paraffin as much as possible. For yolky 

 embryos and larvae, like those of Solaster, the mixture of corrosive 

 sublimate and acetic acid, in spite of its disadvantages, will probably 

 give the best results on the whole. 



For making whole mounts other means must be adopted. Good 



