558 CYTOCHEMICAL TESTS ON EMBRYOS 



c. After 12 to 15 minutes in the above mixture, add 0.2 cc. of 2'jt NaOBr, 

 and stir well for 5 minutes. This solution should be freshly made up 

 by pouring 0.7 cc. of liquid bromine into 100 cc. of 5^ NaOH, agitat- 

 ing, and cooling. 



d. Add 0.2 cc. of kOf, urea, stir. 



e. Add 0.2 cc. of 2^ NaOBr and again stir well. The color will now 

 develop if arginine ia present and should attain its maximum intensity 

 in about 5 to 5 minutes. 



The color can be somewhat stabollzed by passing the tissue through h changes 

 of pure glycerine. 



Serra claims that if the tissue is placed In pure NaOBr solution for 

 5 minutes after step "e" (above) the color becomes more intense and is 

 stabilized in glycerine. He uses this test for basic proteins, for proteins 

 in general which contain arginine, and for guanldine derivatives in which 

 only one H-atom of one amino group is substituted by a radical of the alkyl 

 or fatty acid type. 



b. THE METHOD OF THOMAS {I9t6) 



With this method the red color develops when a solution of arginine or 

 a protein solution containing arginine is treated with a-naphthol, alkali 

 and hypochlorite. Guanidine itself is negative but when one of the guanidine 

 H-atoma is substituted by an alkyl, fatty acid, or cyano radical, then there 

 is a positive color reaction. The color develops and fades rapidly but the 

 addition of urea helps to hold the color for several minutes. Bapid dehy- 

 dration is critically important, and is accomplished by tertiary butyl 

 alcohol followed by aniline oil. 



a. Fix tissues in Bouin'a fluid. Carnoy's or 10^ formalin are equally 

 good. 



b. Sectioned material must be firmly affixed to slides, with the usual 

 paraffin albumin method. The slides may be left in 70^ alcohol until 

 ready for the tests. 



c. Hydrate the tissues down to distilled water, then place the slide in 

 a-naphthol in 10^ alcohol, by volume. 



d. Transfer to sodium hypochlorite solution for 20 seconds. This is a 

 0.15 normal solution of sodium hypochlorite in 0.05 normal sodium 

 hydroxide. This solution can be made from commercial "Clorox" (see 

 Albanese & Frankston, I9U5) or by the method of Van Slyke & Hi Her 



(1955). 



e. Transfer to urea for 5 seconds. This is an alkaline urea made up by 

 adding 20^ urea to 0.05 normal sodium hydroxide. 



f. Dehydration: 80^ tertiary butyl alcohol for 50 seconds. To the BO cc. 

 of tertiary butyl alcohol add 1 cc. of 5 normal sodium hydroxide and 

 19 cc. of distilled water. Transfer to 100^ tertiary butyl alcohol 

 for 2 minutes. 



Amblystoma testes, Bouin fixation showing positive Arginine reaction. 

 (Courtesy L. JE. Thomas 191+6: Jour. Cell. & Comp. Phyelol. 28:11+5) 



