CYTOCHEMICAL TESTS ON EMBRYOS 



555 



H 



OH ,0(P05%) 



Guanln - d - ribose nucleotid 

 (Ribosnucleic acid) 



The distribution of ribonucleic acid at 

 three stages in the oogenesis of the 

 amphibian egg. (Redrawn from Brachet, 

 1944.) 



OlPOj^) 



Guanln - 2-desoxy - d - ribose - 

 nucleotlc (Thymonucleic acid) 



IDENTIFICATION OF NUCLEIC ACID BY TEE USE OF ENZYMES :* 



1. Rlbonuclease (crystalline) can be used to digest away the ribonucleic acid, 

 following which Toluldine Blue will give blue nuclei and cytoplasm colorless 

 or following which Unna's stain would give green nuclei and colorless cyto- 

 plasm. See Kunltz (19'+0) for simple method of preparing crystalline rlbon- 

 nuclease. 



A concentration of 0.1 mgm./ cc. of water, properly buffered 

 (Mcllvalnes buffer to pH 7.0), of the crystalline rlbonuclease should be 

 used with the tissue. Incubated at 50OC. for 5 hours. Wash with distilled 

 water; stain with Toluldine Blue or Unna. The best fixatives for this test 

 are Camoy, or the a Icohol-formol- acetic mixtures, Zenker's without formol 

 but with acetic acid. Fixation no more than 1 hour for Amphibian eggs. 



A positive stain following this procedure identifies thymonucleic acid, 

 and will be essentially nuclear. 



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2. Thymonuclease (McCarty, I9I+6: Jour. Gen, Physiol. 29:125) acts In a few 

 minutes at room temperature. If the tissues are rinsed and followed with 

 Toluldine Blue or Unna, the positively staining elements will represent the 

 ribonucleic acid which is essentially nucleolar and cytoplasmic. (It must 

 be remembered, however, that the specificity of these enzymes has not been 

 conclusively demonstrated, and certain variables are Involved in their use.) 

 Brachet {Private Com.) reports that Miss MacDougall at Cold Spring Harbor 

 stated that she obtained rlbonuclease completely free of proteolytic action 

 and that it worked Just the same on sections. Brachet was unable to find a 

 decrease In the arglnine and pyronlne reaction after digesting tissues with 

 rlbonuclease. 



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♦These enzymes are still very expensive. 



