58 MICROSCOPIC TECHNIQUES 



Thomas Method for Arginine and Arginine-Containing 

 Proteins 



SPECIAL REAGENTS 



0.1% a-Naphthol in 10% (by vol.) Ethyl Alcohol. 



0.15 N Sodium Hyp/ochlorite in 0.05 N Sodium Hydroxide. For 

 preparation of the hypochlorite see page 239; or prepare from 

 Clorox which is standardized and then stored at 3-5° in a dark 

 bottle. Dilute the hypochlorite to the proper strength each time 

 before use. Clorox is approximately 1.6 A^; standardize by adding 

 1 ml. Clorox to 5 ml. 1 A^ potassium iodide, 8 ml. cone, hydro- 

 chloric acid (sp. gr. 1.19), and 45 ml. water. Titrate with 0.1 N 

 sodium thiosulfate using starch indicator. 



20% Urea in 0.05 N Sodium Hydroxide. 



Tertiary Butyl Alcohol Solution. Add 1 ml. 5 N sodium hydroxide 

 and 19 ml. water to 80 ml. of the tertiary butyl alcohol. Shake 

 well and let stand; an aqueous layer collects on the bottom of the 

 vessel. 



Pure Tertiary Butyl Alcohol. 



Aniline. 



Toluene. 



PROCEDURE 



1. Fix animal tissues in Bouin fluid (75 ml. saturated picric 

 acid soln., 25 ml. formalin, 5 ml. glacial acetic acid) . Onion root 

 tips were treated with medium chrome-acetic fixative. 



2. Prepare paraffin sections in the usual manner. Do not remove 

 paraffin from sections until test is to be applied. 



3. Place slide with sections in the a-naphthol soln. for at least 

 3 min. 



4. Transfer to each of the following solns. in succession for the 

 periods indicated: hypochlorite, 20 sec; urea, 5 sec; 80% tertiary 

 butyl alcohol, 30 sec; pure tertiary butyl alcohol, 2 min.; aniline, 

 2 min.; toluene, 5 sec; and finally mount in Clarite. Use a stop watch 

 to time the immersions in each fluid. 



TRYPTOPHANE IN PROTEINS 



The red or violet color formed with proteins in the presence of 

 phosphoric acid is the basis of the Romieu reaction. Blanchetiere 



