210 RADIATION BIOLOGY 



coagulu or i)iecipitates of the substrates. This suggests that the sub- 

 strates \\ liich are colored red have bound the dye in such steric relation 

 that the dimor and polymer association occur, while those colored blue 

 have the dyr molecules more widely separated so that substantially all the 

 color is due to the blue monomer. According to this interpretation, the 

 color of the dye is in effect a reflection of the intramolecular structure of 

 the substrate. The polysaccharide sulfuric esters (e.g., chondroitin sul- 

 fate of connective tissue) are decidedly metachromatic. A careful spec- 

 trophotometric analysis has shown that a metachromasia distinguishes 

 RXA from DNA, a difference which presumably is related to the highly 

 branched structure of the former (Flax and Himes, 1950, 1952). 



Methyl green basophilia is another staining reaction which appears 

 to reflect the intramolecular configuration of the substrate. This dye 

 stains normal DNA; with i-are exceptions it does not stain UNA. In 

 vitro the formation of the salt methyl green-deoxyribonucleate is depend- 

 ent on the nucleic acid being in a state which forms highly \'iscous solu- 

 tions (Kurnick, 1947, 1949; Kurnick and Mirsky, 1949), and therefore 

 reduction or loss of methyl green basophilia of nuclei has been interpreted 

 as evidence of a physical change in the DNA molecule which is similar to 

 that which is accompanied by loss of viscosity of solutions of the acid, 

 a change which is usually called " depolymerization " (Pollister and 

 Leuchtenberger, 1949; Leuchtenberger, 1950; Leuchtenberger e/ a/., 1949; 

 Harrington and Koza, 1951). Such changes in methyl green basophilia of 

 nuclei have been noted to result from experimental treatment (heat, 

 deoxyribonuclease digestion, ionizing radiation) and also to accompany 

 pathological nuclear degeneration. 



3-2a. Nucleal Reaction. Goldschmidt (1904) and the other adherents 

 of the views embodied in what was called the "chromidial hypothesis" 

 believed that they had in basophilia a sort of qualitative test for chro- 

 matin by which they could detect this substance even after its extrusion 

 from the nuclei into the cytoplasm. This was an over-optimistic point of 

 view and led to widespread distrust of cytochemical conclusions from 

 staining I'esults. Feulgen and Uossenbeck (1924) developed a specific 

 cytochemical test foi' chromatin (Fig. i\-'2F) which not onl,y finally dis- 

 posed of the chromidial hypothesis, sensu strictu, but also led to great 

 strides in clarification of the whole problem of intracellular distribution of 

 luicleic acids. For nearly thirty years two different nucleic acids had 

 been recognized by chemists. One, obtainable in (juantity from yeast 

 and often called yeast nucleic acid, had been shown to contain a pentose; 

 the other, identical with the acidic component of Miescher's iniclein but 

 usually later obtained from the thymus gland, contained a sugar that was 

 clearly not a pentose and was generally considered to be sort of hexose 

 (see Le\'ene and Bass, 1931; Dax'idson, 1950). Feulgen and Rossenbcck 

 discovered that mild acid hydrolysis, which was known to split off the 



