3W 



CYTOCHEMICAL TESTS ON EMBRYOS 



A modified Mlllon's reaction, devised by Serra and Lopes (19^5) lias 

 been used to detect the presence of tyrosine, in tiie protein molecule, but 

 the color Is also produced by other phenolic compounds. A transient color 

 is produced by tryptophane but the tyrosine color attains Its maximum In- 

 tensity in about 5 minutes and lasts for some months, fading gradually with 

 time. Their procedure follows: 



a. Immerse tissues in mercuric solution for 30 minutes. This la made up 

 of 7.5 gms. BgSOjj.; 5.5 gms. HgClg; and 7.O gms. NagSOl^ dissolved In 

 85 cc. of distilled water to which 12.5 gnis. of cone. :^SOi+ had been 

 added, the whole being made up to 100 cc. volume with distilled water. 

 The reaction should take place in a glass stoppered bottle in water 

 bath at 60OC. 



b. Cool the bottle in running water for 10 minutes. 



c. Add to the mercuric solution in the bottle an equal volume of distilled 

 water. 



d. Develop the color by adding a few drops of -1 M NaNC^ (6.9^ aqueous), 

 freshly prepared. The color may last for months if the tissues are 

 mounted in glycerine. Compress cells apart beneath coverslip. 



A second procedure may be used and is known as the diazo reaction for 

 hlstidine and tyrosine. This test gives an orange or yellow color in the 

 presence of hlstidine and tyrosine of the proteins. It is the hlstidine 

 which imparts the reddish tinge. Lison (I956) follows a different procedure 

 to demonstrate the presence of phenolic compounds and axoproteids. Serra' s 

 (19't-6) procedure follows: 



a. Treat tissues for 2 to 3 minutes with saturated aqueous solution of 

 sodium carbonate. 



b. Add a few drops of diazo reagent, stir well, and observe in pure 

 glycerine. 



The diazo-reagent (prepare Just before using, keep cool) 



Place 1.5 cc. of sulphanlllc acid In a 50 cc. flask in an Ice 



bath. 



This Is made by dissolving O.9 gms. pure sulphanlllc acid 



in 9 °c« of concentrated HCl, and adding water to make 100 cc. 



Add 1.5 cc. of a 51^ aqueous solution of NaNO^, shaking well. 



Add 6 cc. of NaNOg after 5 minutes, constantly shaking mixture. 



Add cooled distilled water to make 50 cc. total volume. 



********^*** 



Nuclei of 

 spermatozoa 



vH>:^ 



Tails of 

 spermatozoa 



L 



J 



Modification of the Sakaguchi 

 test for Guanldine derivatives. 

 Trlturus vulgaris teatls. 



Courtesy J. R. Baker, I9U7: Quart. Jour. 

 Microsc. Science. 88:115. 



