556 CYTOCHEMICAL TESTS ON EMBRYOS 



NOTE: The student ia advised to run controls for all of the preceding page proce- 

 dures, and to gain a prior acquaintance with the standard procedures with 

 safranln-llght -green and the iron haematoxylln. It must be remembered that 

 the chromosomes consist of more than thymonucleic acid; that both types of 

 nucleic acids may be found in both the cytoplasm and the nucleus; that stain- 

 ing reactions which are essentially chemical reactions involving the use of 

 enzymes rather than adherent staining, depend on fixation, pH, and tempera- 

 ture, and concentration of the enzymes (Stowell and Zorzoll, 19'+7); and that 

 the technique of combining the use of enzymes with specific staining proce- 

 dures has not yet been fully checked for reliability. 



Another approach which, if combined with the above, would give more re- 

 liable results, la the U.V. absorption photography ( Caspersson, Lavin and 

 others). Both nucleic acids absorb in the ultraviolet range, but the bands 

 are sufficiently far apart so that if the tissues are pre-treated with 

 enzymes, then the absorption Is read, an analysis can be made. 



F. GENERAL PBOTEIN TESTS ( STAHDARD) : 



1. Biuret Test for Peptides - this ia a crude and relatively Insensitive teat 

 for peptides or proteins in general, but has the advantage of being rapid 

 and rather simple. The procedure is aa follows: 



a. Harden tissues In 10^ formaldehyde for 2k hours if formalin is not 

 Included in the fixative. Wash thoroughly- 



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

 reactions must be carried on in an alkaline environment which tends 

 to macerate the tlaauea. It ia a more satisfactory test for the pre- 

 sence f^f these substances in relatively large pieces of tissue. 



c. Place the tissue In 1^ NaOH or 1^ KOH in a watchglaas; add a few drops 

 of 1^ aqueous CuSOi^, and stir. A red color develops in the presence 

 of simple peptides: a blue-violet color with the higher peptide and 

 proteins. 



2. Xanthoproteic Beaction - 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 tissuas in 10^ formaldehyde for 24 hours, if formalin is 

 not included In the fixative. Strong fixation is necessary. 



b. Immerae the tissue in concentrated HNO^ for some minutes, until it be- 

 comes 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 teat. 



e. Mount directly and examine in pure glycerine. 



5 . Ninhydrin Beaction for Amino Acids and Lower Peptides - this test glvea a 

 blue or violet color in the presence of amino acids, free or bound peptides 

 and proteins. The reaction ia not highly specific, however, for it is 

 negative to amino acids proline and hydroxyproline, and it Is positive to 

 certain amines, aldehydes, augars with free aldehyde or keto groups and 

 ammonia compounds. With these non-protein and non-amlno acid compounds, 

 the color reaction is much less intense and tends to be reddish instead of 

 blue. 



Serra (19'+6) 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 aa follows (Serra and Lopea, 19'+5)*. 



a. Sectioned material Is immersed in equal volumes of O.U^ solution 



triketohydrinden-hydrate (ninhydrin) in distilled water and any phos- 

 phate buffer held to pH 6.98. The phosphate buffer may be made by 

 adding 6 cc. of a M/15 secondary sodium phosphate to ^4. cc. of M/ 15 

 primary potassium phosphate. 



