SECTION TWO 



B. Carbol thionin (NicoUe). 



C. Picric acid i% aqueous. 



Technique: 



1. Formalin-fixed specimens are placed in a large volume of 

 Solution A, which is changed once or twice a day. The time 

 required for complete decalcification will depend, of course, on the 

 thickness and the nature of the specimen. The bones of young 

 animals usually take from twenty-four to forty-eight hours, but in 

 other cases as long as a week may be necessary. Decalcification is 

 complete when the bone has become flexible and can easily be 

 punctured with a needle. 



2. Make Celloidin or frozen sections. 



3. Rinse in water for ten minutes. 



4. Stain with Solution B for 10 minutes; then rinse in distilled 

 water. 



5. Immerse in Solution C for half to one minute; then wash in 

 water. 



6. Differentiate with 70% alcohol for five to ten minutes until 

 the stain ceases to come out of the sections. 



7. Dehydrate with 96% alcohol; clear in origanum oil; then 

 mount in balsam. 



Results: 



Ground substance, yellow to brown; bone canaliculi, dark 

 brown to black; cells, red ; ground substance of cartilage, brilliant 

 purple. 



Note: Carbol thionin (Nicolle) deteriorates after a few weeks 

 and this stain is therefore best when freshly prepared. 



References : 

 Carleton, H. M. & Leach, E. H. (1947), pp. 213-214. 

 Schmorl, G. (1928). 



CARMINE - METHYLENE BLUE (Schultz Stain) 

 For demonstrating sodium urate in animal tissue 



Solutions required: 



A. Distilled water . . . . . . 64 ml. 



Lithium carbonate . . . . 0-5 gm. 



163 



