THE MITOTIC CYCLE 



final results depend upon the nature of the fixative which was used, 

 and conclude that 'with our present limited knowledge and technics, 

 it would seem advisable to use the ribonuclease technic only crudely 

 to confirm observations made by other methods'. Davidson^'' believes 

 that the main difficulty resides in the contamination even of the 

 crystalline enzymes with traces of proteolytic ferments*. It has been 

 suggested that certain inorganic reagents can remove ribonucleic acid 

 from tissue sections more effectively than the enzyme. Erickson et 

 alii^^ suggest perchloric acid for this purpose, while Vendrely- 

 Randavel^*^ and Ponyet^^ advise the use of normal hydrochloric acid 

 for ten minutes. 



This difficulty of proteolytic contamination is even more marked 

 with deoxyribonuclease, which does not share the heat-stable nature of 

 the former enzyme, thanks to which it can be mainly freed of impurities. 

 Several workers have added a very small amount of gelatin to their 

 preparations of deoxyribonuclease which they have used for the diges- 

 tion of sectioned biological material; Webb and Jacobson^^ have used 

 small concentrations of tryptic inhibitors in the digestion mixture. 



The extent to which deoxyribonucleic acid can be removed from the 

 nucleus by digestion again depends among other factors upon the 

 fixative originally used. Formalin should be avoided (Stowell,*^ 

 Sanders^*); chilled acetone is recommended by Davidson.^^ Nuclei 

 in the digested sections are sometimes still faintly Feulgen-positive 

 (Stowell, Davidson, op. cit.), although a completely negative reaction 

 following digestion is reported by Catcheside and Holmes^^ in the 

 bands of salivary chromosomes, and by Webb and Jacobson®^ in smears 

 of mouse and human cells. Catcheside and Holmes find that thymonu- 

 clease prepared from the spleen does not act on bean-root nuclei unless 

 ribonuclease is used at the same time, while pancreatic deoxynuclease 

 is effective alone. 



Danielli^' has criticized the use of these enzymes in cytochemistry 

 with some severity, on the grounds of their probable impurity. He 

 further suggests that a nuclease entirely free of proteolytic activity 

 might fail to act if even a monolayer of protein surrounded the sub- 

 strate within the tissue section. It is possible, however, that the recent 

 successful use of nucleases in the presence of proteolytic inhibitors by 

 Webb and Jacobson®^ have diminished the force of these criticisms. 



But for the use of ribonuclease in cytochemistry there would have 

 been no advance in the microscopical identification of ribonucleic 

 acid subsequent to the recognition of its affinity for basic stains. Why 

 such dyes as methylene and toluidine blue should be adsorbed more 

 readily by RNA than by DNA is still not known, though it is surmised 

 that free phosphate groups at the surface of the molecule are more 



* McDonald* has prepared ribonuclease free of all proteolytic activity. 



ID 



