330 CYTOCHEMICAL TESTS ON EMBRYOS 



h. Xylene, 2 changes of 5 minutes each. Wipe forceps dry before carrying 



another slide through procedure from stain so that staining solution will not 

 be contaminated. 



i. Mount slide in xylene-balsam medium. 



6. Ribonuclease test : 



a. Hydrate 2 (a + b) slides froin same source to distilled water as in 5. a. above 



(1) and (2). 



b. (1) Enzyme reaction: 0.01% ribonuclease in distilled water, room tempera- 



ture for 1 hour. Purified ribonuclease is obtained from Worthington Bio- 

 chemical Corp. , Freehold, N. J. 



(2) Solvent control: Distilled water, room temperature 1 hr. 



c. Stain slides a and b at same time as in section 5, b to i. 



G. Identification of Nucleic Acid by the Use of Enzymes:* 



1. Ribonuclease (crystalline) can be used to digest away the ribonucleic acid, fol- 

 lowing which Toluidine Blue will give blue nuclei and cytoplasm colorless or fol- 

 lowing which Unna's stain would give green nuclei and colorless cytoplasm. See 

 Kunitz (1940) for simple method of preparing crystalline ribo-nuclease. 



A concentration of 0. 1 mgm. /cc. of water, properly buffered (McUvaines buffer 

 to pH 7. 0), of the crystalline ribonuclease should be used with the tissue, incu- 

 bated at BO'-'C. for 3 hours. Wash with distilled water; stain with Toluidine Blue 

 or Unna. The best fixatives for this test are Carnoy, or the alcohol-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. 



2. Thymonuclease (McCarty, 1946: Jour. Gen. Physiol. 29:123) acts in a few min- 

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

 Blue or Unna, the positively staining elements will represent the ribonucleic acid 

 which is essentially nucleolar and cytoplasmic. (It must be remembered, how- 

 ever, 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 ribonuclease 

 completely free of proteolytic action and that it worked just the same on sections. 

 Brachet was unable to find a decrease in the arginine and pyronine reaction after 

 digesting tissues with ribonuclease. 



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

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

 safranin-light-green and the iron haematoxylin. It must be remembered 

 that the chromosomes consist of nnore than thymonucleic acid; that both 

 types of nucleic acids may be found in both the cytoplasm and the nucleus; 

 that staining reactions which are essentially chemical reactions involving 

 the use of enzymes rather than adherent staining, depend on fixation, pH, 

 and temperature, and concentration of the enzymes (Stowell and Zorzoli, 

 1947); and that the technique of combining the use of enzymes with specific 

 staining procedures has not yet been fully checked for reliability. 



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

 liable results, is 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, in analysis can be made. 



♦These enzymes are still very expensive. 



