232 DYEING 



above that of water. Ground cytoplasm commonly gives figures in 

 the neighbourhood of 1-353, but a less aqueous object like the mito- 

 chrondrial Nehenkern of an insect spermatid gives 1-376.^^^ When 

 fixation takes place, the water-relations of the protein chains are 

 entirely changed, and in a balsam preparation there is no water left. 

 The protein has now a refractive index of about 1-52 to 1-54. It is 

 precisely for this reason that we use Canada balsam as a mounting 

 medium: it has almost the same refractive index as the 'dry' pro- 

 tein, and the latter is therefore transparent. The spaces between 

 the protein fibres are now filled with balsam. If there was a lot of 

 water associated with the protein in life, these fibres will be far 

 apart; if there was little water, they will be close, so that there 

 will be more fixed tissue per unit volume. In a word, the tissue will 

 be dense in the strict physical sense. 



Let us now imagine the dyeing of two fixed tissue-constituents, 

 the one consisting of a greater length of protein chain per unit 

 volume than the other (and therefore denser), but both exactly 

 equal in the number of acidic and basic groups per unit length of 

 protein chain. Any dye, whether basic or acid, will necessarily be 

 taken up in greater quantity by the former, w^hich will appear 

 darker in the finished preparation. There will, however, be no 

 obvious indication of the cause of the uptake of more dye. It 

 might equally well have been due to an entirely diflferent cause: 

 not to any difference in density, but to the fact that the protein was 

 (for instance) particularly acidic, and therefore bound to a lot of 

 basic dye. 



Metaphase chromosomes are genuinely denser than the sur- 

 rounding cytoplasm, and the depth of their colouring is partly 

 due to this. One would expect them to take up basic dyes strongly, 

 but the protein constituent of the nucleoprotein can also be much 

 more strongly coloured than the cytoplasm by acid dyes. In the 

 same way the chromatin of the interphase nucleus can be much 

 more deeply coloured than the nuclear sap by acid dyes. Indeed, 

 deliberate use is made of acid dyes to show chromatin in certain 

 techniques. It has already been mentioned (p. 205) that acid 

 fuchsine was used for this purpose by Mallory,^^^ and azocarmine 

 by Heidenhain.^^^ The colouring of the protein of chromatin by 

 acid dyes is made easier if the nucleic acids are first eliminated by 

 enzyme action. Ribonuclease and DNA-ase both help the sub- 

 sequent action of acid dyes in colouring the protein of nucleo- 

 protein. ^^^ 



