CYTOCHEMICAL TESTS ON EMBRYOS 337 



b. Place the material in a watchglasa and over a 'boiling water tath, 

 among the vapors, for 1 to 2 minutes after the water 1)0118. 



c. Mount in pure glycerine. If the- tissues are thick, compress teneath 

 a coverslip to separate the cells from each other. The color will 

 fade within a few hours. 



G. TESTS FOB VABIOUS COMMON AMINO ACIDS : 



1. Arginine : The development of histo-chemlcal teats of great specificity has 

 immeasurahle significance in relation to an understanding of cell morphology 

 and physiology, particularly in respect to the nuclear inclusions. Thomas 

 (191+6) and Serra (19^+6) following Sakaguchi (I925) have perfected the test 

 for arglnlne. 



The enjjirical formula for arginine is 

 H3N NH 

 \/ 



/ 



OGH 



N]^ 



According to Thomas {19'+6) "when the Sakaguchi reaction is applied to 

 a protein, a color is Imparted to the protein molecule." Ejy the methods of 

 Thomas and of Serra, the red color of the following reactions may he re- 

 garded 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 intermltotic chromatin than in the remainder of 

 the cell. The spermatozoan heads, representing concentrated nuclear 

 material, give a most intense reaction (see Thomas, 19'+6). 



Thomas (I9U6) and Serra (191*6) have independently modified the original 

 Sakaguchi (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 transient but can be prolonged for several hours by 

 proper dehydration. The ufjual ethyl alcohol tends to extract some of the 

 color, as do most of the other dehydrants, but Thomas il9k6) 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 (19'+6) now advises the use of glycerine in which the color seema 

 to be stabilized for many months. 



a. THE METHOD OF SERRA (!946) 



a. Harden fixed tissues in 10^ formaldehyde for 2U hours unless formalin 

 was included in the original fixative. 



b. Prepare an alkaline a-naphthol-urea mixrixre and bring it to 0° to 5°C., 

 in a watchglass. The mixture is as follows: 



1. 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 1+0^ alcohol. 



2. O.'? cc. normal NaOH. 



5. 0.2 cc. of hCfjii aqueous urea solution. 



