Stains and Staining 97 



preserved in 70 per cent alcohol made slightly alkaline by saturating it 

 with borax. When they are thoroughly hardened, a 0.5 per cent solu- 

 tion of alizarin red S in absolute alcohol is added in the proportion of 

 1 ml of stain for each 100 ml of preservative. The alizarin forms a red 

 lake with the calcium in the bones. When the bones are red enough, the 

 surplus stain is washed out of the other tissues with alkaline alcohol. 

 The embryos are then dehydrated and mounted in balsam. 



Cartilaginous skeletons cannot be directly stained, as are bones, but 

 may be indirectly stained in toluidine blue. The embryos are fixed in any 

 fixative not containing picric acid— mercuric mixtures are preferred by 

 most people— and thoroughly washed. They are soaked for 24 hr in: 



Van Wijhe's Stain: 



70% alcohol 100 ml 



Hydrochloric acid 0.1 ml 



Toluidine blue 0.1 g 



Then they are differentiated in 0.1 per cent hydrochloric acid in 70 

 per cent alcohol until no more color comes away. Dehydration and mount- 

 ing in balsam will show the cartilage alone stained clear blue. 



