CYTOCHEMICAL TESTS ON EMBRYOS 



333 



The empirical formula for arginine is 

 H^Ns yNH 



N 

 /^ OOH 



NH- 



According to Thomas (1946) "when the Sakaguchi reaction is applied to a protein, 

 a color is imparted to the protein molecule. " By the methods of Thomas and of 

 Serra, the red color of the following reactions may be regarded as proof-positive 

 of the presence of arginine. 



It is suggested that sections of the testes and of the ovary be tested for arginine. 

 The red color generally develops in both the cytoplasm and the nucleus of most 

 cells, but there is considerably more color in the chromosomes, nucleoli, and 

 intermitotic chromatin than in the remainder of the cell. The spermatozoan 

 heads, representing concentrated nuclear material, give a most intense reaction 

 (see Thomas, 1946). 



Thomas (1946) and Serra (1946) have independently modified the original Saka- 

 guchi (1925) reaction as a specific test for arginine in biological materials. The 

 Thomas procedure is negative for guanidine, urea, creatine, creatinine, and 

 other amino acids that might be encountered in biological tissues. The presence 

 of arginine is demonstrated by a strong red or red-orange color which is tran- 

 sient but can be prolonged for several hours by proper dehydration. The usual 

 ethyl alcohol tends to extract some of the color, as do most of the other dehy- 

 drants, but Thomas (1946) has found that tertiary butyl alcohol will dehydrate 

 the tissues without the removal of color and aniline oil is used to clear. The 

 system must be kept alkaline because the color fades in either neutral or acid 

 media. 



Serra (1946) now advises the use of glycerine in which the color seems to be sta- 

 bilized for many months. 



a. The Method of Serra (1946) 



(1) Harden fixed tissues in 10% formaldehyde for 24 hours unless formalin 

 was included in the original fixative. 



(2) Prepare an alkaline a-naphthol-ur ea mixture and bring it to 0° to 5°C. , 

 in a watchglass. The mixture is as follows: 



(a) 0. 5 cc. diluted a-naphthol. Use stock solution 1% crystallized 

 a-naphthol in 96% alcohol and, just before using, dilute to 1/10 with 

 40% alcohol. 



(b) 0. 5 cc. normal NaOH. 



(c) 0. 2 cc. of 40% aqueous urea solution. 



(3) After 12 to 15 minutes in the above mixture, add 0. 2 cc. of 2% NaOBr, 

 and stir well for 3 minutes. This solution should be freshly made up by 

 pouring 0. 7 cc. of liquid bromine into 100 cc. of 5% NaOH, agitating, 

 and cooling. 



(4) Add 0. 2 cc. of 40% urea, stir. 



(5) Add 0. 2 cc. of 2% NaOBr and again stir well. The color will now develop 

 if arginine is present and should attain its maximum intensity in about 3 

 to 5 minutes 



The color can be somewhat stabilized by passing the tissue through 4 changes 

 of pure glycerine. 



