42 MICROSCOPIC TECHNIQUES 



CAROTENE, CAROTENOIDS, AND VITAMIN A 



The solubility of carotene, carotenoids, and vitamin A in organic 

 solvents makes it necessary to employ frozen sections of tissue for 

 histological studies on these substances. Unstained sections show 

 yellow, orange, or brown regions due to the presence of constituents 

 of this nature. The blue coloration given by concentrated sulfuric 

 acid with these compounds has been employed by Steiger (1941) 

 for the demonstration of carotene in leaves. The deep violet color 

 developed in the presence of aqueous 1% iodine in 7% potassium 

 iodide (Lison, 1936, page 245) is also characteristic of these 

 polyenes, and when treated with oxidizing agents, such as chromic 

 acid, they are bleached. Bourne (1935) adapted the Carr-Price 

 reaction to tissue sections by placing frozen sections directly into 

 a chloroform solution of antimony trichloride. It is well known 

 that the blue color due to vitamin A fades very rapidly, while that 

 due to carotene persists. As shown by Raoul and Meunier (1939), 

 sterols produce a red color in the Carr-Price test. The detection uf 

 vitamin A in tissue by fluorescence is described on page 104. 



Steiger Method for Carotene in Leaves 

 SPECIAL REAGENTS 



Alkali-Alcohol Mixture. Combine 1 vol. saturated potassium hy- 

 droxide with 2 vol. of 40% alcohol and 3 vol. tap water. 

 Concentrated Sulfuric Acid. 



PROCEDURE 



1. Place green leaves in the alkali-alcohol mixture in a wide- 

 mouth bottle and seal the glass stopper with vaseline. 



2. After several days in the dark, when the fluid is green and 

 the tissue yellow, transfer to distilled water for several hr. 



3. Place small pieces of tissue on a slide and dry with filter paper. 



4. Add 1 drop cone, sulfuric acid. 



Result. Carotene is indicated by the appearance of dark blue 

 crystals visible under the microscope. Grossly, a green color chang- 

 ing to blue can be observed. 



