334 



CYTOCHEMICAL TESTS ON EMBRYOS 



Serra claims that if the tissue is placed in pure NaOBr solution for 3 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 guanidine 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 (1946) 



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

 tein solution containing arginine is treated with a-naphthol, alkali and hypo- 

 chlorite. Guanidine itself is negative but when one of the guanidine H-atoms 

 is substituted by an alkyl, fatty acid, or cyano radical, then there is a pos- 

 itive color reaction. The color develops and fades rapidly but the addition of 

 urea helps to hold the color for several minutes. Rapid dehydration is crit- 

 ically important, and is accomplished by tertiary butyl alcohol followed by 

 aniline oil. 



(1) Fix tissues in Bouin's fluid. Carnoy's or 10% formalin are equally good. 



(2) Sectioned material must be firmly affixed to slides, with the usual paraf- 

 fin albumin method. The slides may be left in 7 0% alcohol until ready for 

 the tests. 



(3) Hydrate the tissues down to distilled water, then place the slide in a-naph- 

 thol in 10% alcohol, by volume. 



(4) 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, 1945) or by the method of Van Slyke & Hiller (1933). 



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

 adding 20% urea to 0. 05 normal sodium hydroxide. 



(6) Dehydration: 80% tertiary butyl alcohol for 30 seconds. To the 80 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. E. Thomas 1946: Jour. Cell. G Conip. Physiol. 28:145) 



(7) Clearing: 



(8) Mounting: 



100% aniline oil for 2 minutes. 

 100% toluene for 5 seconds. 

 Clarite. 



Tyrosine : According to Serra (1946) "Tyrosine seems to be present in almost 



all natural proteins in amounts which are not very different for the various 



classes, excepting principally silk fibroin, pepsin and insulin. " The formula 



for tyrosine is 



OOH 



HO 



NH- 



