62 



HEWSON SWIFT 



500 



cartilagt matrix 



700 500 



Wavelength 



600 



700 



Fig. 3. Metachroraasy of azure B in acetic alcohol-fixed salamander {Ambystoma) 

 tissues. Absorption curves run with microphotometer on sections 8m thick. DNA 

 from nucleus after ribonuclease, showing predominant blue-green orthochromatic 

 peak; PNA from Nissl substance, showing purple metachromatic peaks; mucin and 

 cartilage matrix showing reddish PNA metachromatic peaks markedly different 

 from PNA. Dye concentration 0.2 mg./ml. at pH 4.0; stained for 2 hours at 40°. [From 

 Flax and Himes^*.] 



distinguish basophilia of sulfuric esters from that of nucleic acid phosphoryl 

 groups. Absorption curves for azure B, made on small regions of tissue with 

 a microphotometer by Flax and Himes^* are shown in Fig. 3. The curves 

 for cartilage matrix and goblet cell mucin are markedly different than those 

 of nucleic acids. 



Under appropriate staining conditions nucleic acid may also show meta- 

 chromatic staining. ^^-^^ Further, Flax and Himes^* demonstrated that the 

 conditions may be so adjusted that DNA stains mainly orthochromatically, 

 and PNA metachromatically, so that DNA is colored a blue-green, cyto- 

 plasmic and nucleolar PNA a red-purple. Absorption curves for DNA and 

 PNA staining are shown in Fig. 3. 



In general, conditions which favor increased nucleic acid binding, such 

 as increasing pH and dye concentration, or removal of amino groups mth 

 nitrous acid, favor increase in the dimer or polymer peaks. With a decrease 

 in binding, brought out by lowering pH and concentration, and partially 

 blocking phosphoryl groups with chromic acid, the monomer peak is more 

 prominent. This would support the concept that dye interaction and con- 

 sequent color change takes place after binding, and for structural reasons 

 occurs more readily with PNA than DNA. It is also possible, though prob- 

 ably less likely, that dye intraction occurs before binding, and that PNA 

 has a higher affinity for polymers and DNA for monomers. Flax and 

 Rimes'^* have also showed that, for one set of staining conditions, the shape 

 of the absorption curve of azure B with PNA or DNA is approximately 

 constant whether from regions of light or heavy dye binding. This would 

 imply that polymerization occurs mainly between dye ions bound to the 



" G. B. Wislocki, H. Bunting, and E. W. Dempsey, Am. J. Anat. 81, 1 (1947). 

 " M. Weissman, W. H. Games, P. S. Rubin, and J. Fisher, J. Am. Chem. Soc. 74, 

 1423 (1952). 



