SECTION TWO 



4. Macerate for five to seven days, depending on the size of the 

 animal, in several changes of 2% aqueous potass, hydroxide. 



{Note: This process is hastened by exposure to sunlight.) 



5. Transfer to Solution C for about twenty-four hours when the 

 bones should be well stained. If the specimen has been insuffi- 

 ciently macerated the soft tissue will be sHghtly stained, in which 

 case the specimen may be destained rapidly in acid alcohol (1% 

 sulphuric acid in 95% alcohol). 



6. Dehydrate by leaving the specimen in three changes of 

 cellosolve for six hours in each. Instead of cellosolve, 50%, 80% 

 and 90% alcohol, followed by three changes of benzol may be 

 used for dehydration. Small embryos require less time in the 

 dehydrating fluids. 



7. Clear by transferring to solutions of 25%, 50% and 75% 

 methyl salicylate in cellosolve for twenty-four hours in each. 



8. Store in methyl salicylate. 



Note: If glycerin is used for clearing the technique has to be 

 modified as follows : 



Omit stage 6 and transfer directly from the Alizarin Red S 

 solution into a series of 50%, 70% and 80% glycerin for twenty- 

 four hours in each ; then store in pure glycerin. 



Results: 



Soft tissues: transparent. Osseous tissue: deep blue. Carti- 

 lage : dark blue. 



Note: The relative degree of ossification and chondrogenesis 

 which has taken place is indicated by the intensity of the stains. 

 Bone and cartilage may be stained separately by omitting stage 2, 

 or stage 5 for cartilage. 



Reference: Williams, T. W. (1941). 



For foetal specimens 



The technique is particularly suitable for mammalian embryos, 

 for demonstrating minute bones and foetal ossification. 



127 



