332 CYTOCHEMICAL TESTS ON EMBRYOS 



H. General Protein Tests (Standard); 



1. Biuret Test for Peptides - this is a crude and relatively insensitive test for pep- 

 tides or proteins in general, but has the advantage of being rapid and rather 

 simple. The procedure is as follows: 



a. Harden tissues in 10% formaldehyde for 24 hours if formalin is not included 

 in the fixative. Wash thoroughly. 



b. If the material is to be sectioned, it should be cut thickly. The reactions 

 must be carried on in an alkaline environment which tends to macerate the 

 tissues. It is a more satisfactory test for the presence of these substances 

 in relatively large pieces of tissue. 



c. Place the tissue in 1% NaOH or 1% KOH in a watchglass; add a few drops of 

 1% aqueous CuSO^, and stir. A red color develops in the presence of simple 

 peptides; a blue-violet color with the higher peptide and proteins. 



2. Xanthoproteic Reaction - this is also a crude but simple test for proteins and is 

 positive in the presence of tyrosine, phenylalanine, tryptophane, all phenolic 

 compounds, and all the peptides except the protamines. 



a. Harden the tissues in 10% formaldehyde for 24 hours, if formalin is not in- 

 cluded in the fixative. Strong fixation is necessary. 



b. Immerse the tissue in concentrated HNOo for some minutes, until it becomes 

 intensely yellow. 



c. Wash in distilled water. 



d. Immerse in diluted ammonia, or expose the tissue to ammonia vapors. 

 An orange color indicates a positive test. 



e. Mount directly and examine in pure glycerine. 



3. Ninhydrin Reaction for Amino Acids and Lower Peptides - this test gives a blue 

 or violet color in the presence of amino acids, free or bound peptides and pro- 

 teins. The reaction is not highly specific, however, for it is negative to amino 

 acids proline and hydroxyproline, and it is positive to certain amines, aldehydes, 

 sugars with free aldehyde or keto groups and ammonia compounds. With these 

 non-protein and non-amino acid compounds, the color reaction is much less in- 

 tense and tends to be reddish instead of blue. 



Serra (1946) points out the importance of hard fixation to prevent the color moving 

 about within the tissues and becoming adherent to unnatural cell structures. The 

 reaction is as follows (Serra and Lopes, 1945); 



a. Sectioned material is immersed in equal volumes of 0. 4% solution triketo- 

 hydrinden-hydrate (ninhydrin) in distilled water and any phosphate buffer held 

 to pH 6.98. The phosphate buffer tnay be made by adding 6 cc. of a M/15 

 secondary sodium phosphate to 4 cc. of M/15 primary potassium phosphate. 



b. Place the material in a watchglass and over a boiling water bath, among the 

 vapors, for 1 to 2 minutes after the water boils. 



c. Mount in pure glycerine. If the tissues are thick, compress beneath a cover- 

 slip to separate the cells from each other. The color will fade within a few 

 hours. 



I. Tests for Various Common Amino Acids ; 



1. Arginine : The development of histo-chemical tests of great specificity has im- 

 measurable significance in relation to an understanding of cell morphology and 

 physiology, particularly in respect to the nuclear inclusions. Thomas (1946) and 

 Serra (1946) following Sakaguchi (1925) have perfected the test for arginine. 



