THE DIRECT ATTACHMENT OF DYES TO TISSUES 205 



choose either of these fixatives for work on chromosomes, except in 

 mixtures. 



Despite statements to the contrary, osmium tetroxide allows 

 quite strong colouring of chromatin by basic dyes; but it often 

 makes the nuclear sap basiphil, and this clouds the structure of 

 the nucleus. One would not use this fixative alone in studies of 

 prophase or telophase changes, or in any other investigation re- 

 quiring a glassy nuclear sap. 



The reactions of picric acid with chromatin are very curious. ^^ 

 The protein part is coagulated, but the DNA is set free and dis- 

 solves (compare Levene ^^^). The chromatin is now represented 

 only by protein, and the nature of the latter determines the reaction 

 with dyes. Since the protein component of chromatin is seldom 

 strongly acidic, there is little affinity for basic dyes; but if it 

 chances to be definitely basic (as, for instance, in the spermato- 

 cytes of mammals), acid dyes will be taken up strongly. We thus 

 have the curious spectacle of meiotic chromosomes strongly dyed 

 by acid dyes, but feebly by basic ones. 



It is not unusual for the protein of chromatin to be colourable 

 by acid dyes, after DNA has been separated off by the action of 

 fixatives. Acid dyes are used to colour chromatin in certain tech- 

 niques (acid fuchsine in Mallory's tricolour method ^-^ and azo- 

 carmine in Heidenhain's 'Azan').^^^ After most fixatives the DNA 

 will still be present, but will not mask the colouring of the protein 

 by an acid dye unless a basic dye is used as well. The acid dye 

 works more strongly, however, if DNA-ase is allowed to act fi^rst. 



In the chromatin of the spermatozoa of certain fishes the DNA 

 is combined with protamine. The strongly basic nature of this 

 simple protein, caused largely by its high arginine content, pre- 

 dominates to such an extent that the substance as a whole, with its 

 DNA, is highly acidophil and scarcely takes basic dyes, whatever 

 fixative may have been used. 



If a particular fixative leaves some of the phosphoric groups of 

 DNA still combined with protein, this will necessarily result in a 

 low affinity for basic dyes. 



The reason why formaldehyde interferes with the dyeing 

 of chromatin by borax-carmine follows from the foregoing con- 

 siderations. In alkaline solution carminic acid is an acid dye, with- 

 out affinity for DNA. It can combine, however, with the basic 

 groups of the protein of nucleoprotein, and then, when the tissue is 

 subsequently acidified and it becomes a basic dye, it can fix itself to 



