54 MICROSCOPIC TECHNIQUES 



and softened. The specimen should be pierced or cut to allow escape 

 of carbon dioxide. 



2. If the Diaphanol becomes discolored, transfer to a fresh por- 

 tion of the soln. 



3. Place in 63% alcohol until hardened and then pass through 

 tetralin into paraffin. 



Methods for Staining Chitin 



The softened material, or sections of it, may be tested for chitin 

 by a variety of color reactions. Zander treated for a short time with 

 a drop of fresh iodine in potassium iodide soln., followed by a drop 

 of strong zinc chloride soln. Upon removal of the reagents with 

 water, a violet color is obtained in the presence of chitin. Schulze 

 divided the material into two portions. One was subjected to the 

 procedure of Zander and the other was treated with iodine and then 

 cone, sulfuric acid. The latter test serves to distinguish chitin from 

 cellulose and tunicin since chitin yields a brown color while the 

 others give a blue. Bethe employed freshly prepared 10% aniline 

 hydrochloride containing a drop of cone, hydrochloric acid for each 

 10 ml. After sections were placed in this soln. for 3-4 min., they 

 were rinsed with water and the slides were then placed, sections 

 downward, in a bath of 10% potassium dichromate. Chitin produces 

 a green coloration which becomes blue in tap water or ammoniacal 

 alcohol. 



ASCORBIC ACID 



The stain for ascorbic acid was developed in 1933 by Bourne, 

 who utilized the fact that reduced silver is deposited when ascorbic 

 acid in tissue interacts with acid silver nitrate. Bourne (1936) 

 published a critical survey of this stain and his recommended pro- 

 cedure is given below with a subsequent modification by Barnett 

 and Bourne (1941) designed to increase the specificity of the test 

 by dissolving precipitated silver salts in dilute ammonia. Giroud 

 and Leblond (1936) also investigated the technique and its appli- 

 cations, and in reply to criticisms of the specificity of the stain, 

 these authors ( 1937) point out that the positive test is specific for 

 ascorbic acid but a negative result does not necessarily mean that 



