CYTOCHEMICAL TESTS ON EMBRYOS 337 



Fig. 8. Testis of Rana ridibunda perezi. Arginine reaction (green filter). 



Fig. 9. Spermatozoa of Helix aspersa, heads to upper left. Arginine reaction. 



Fig. 10. Salivary gland chromosomes of Chironomus larvae. Tyrosine reaction. 



Fig. 11. Salivary gland chromosomes of Chironomus larvae. Tryptophane reaction. 



Photographs by courtesy of J. A. Serra 



Since this amino acid results from the hydrolysis of many proteins and the test 

 is sensitive, a satisfactory procedure is available for animal tissues. 



a. Harden the tissues in 10% formaldehyde, if they were not previously fixed 

 in a formalin fixative, for 5 hours; wash well. 



b. Immerse for 3 to 5 seconds in an aqueous solution of sodium silicate. 



c. Immediately immerse the pieces in Voisenet reagent for 10 to 15 minutes, 



in a small glass -stoppered bottle. This reagent is made by adding 1 drop of 

 2% aqueous formol and 1 drop of 0. 15% aqueous NaN02, with stirring, to 10 

 cc. of concentrated HCl. Solution is freshly made before using. 



d. Mount in glycerine and observe directly. Tissues may be compressed apart 

 between coverslip and slide. The color fades hence the tissue must be ex- 

 amined within a few hours. 



J. Test for the -SH Groups : 



The following test gives a stable red coloration in the presence of the tripeptide 

 glutathione. According to Serra (1946): "it is possible not only to demonstrate the 

 existing -SH groups but also to reduce SS groups to SH groups by means of a pre- 

 treatment of the materials with a solution of 10% KCN for 10 minutes. " Of course, 

 the arginine test is also positive for the -SH group proteins. An intense reaction 

 for protein -SH presumably demonstrates the existence of active metabolic and syn- 

 thetic changes in the proteins (Brachet, 1940). Protein denaturation involves an 

 unfolding of polypeptide chains and an increase in -SH reacting groups. Hence, a 

 positive -SH reaction might indicate either an active synthesis or a breakdown of 

 proteins. The procedure follows: 



1. Fix tissues for no more than 4 hours at room temperature in 10% formaldehyde. 

 Rinse in distilled water. 



2. Immerse tissues or sections in 5% aqueous zinc acetate - 30 seconds. Rinse in 

 distilled water. 



3. Treat with 10% aqueous solution sodium nitroprusside containing 2% concentrated 

 ammonia. Brilliant red color develops within 5 minutes. Wash in distilled water 

 and mount in glycerine. 



