INTRODUCTION TO VITAL COLOURING 281 



was certainly seen in the nuclei, but there is every reason to 

 suppose that the chromatin was fixed by the ethanol and then dyed 

 by indigocarmine taken up from the tissue by the fixative. 



The fact that chromatin is usually not coloured may be due in 

 part to the dye having difficulty in penetrating the nuclear mem- 

 brane. It is probable, however, that another cause is more im- 

 portant. The DNA of chromatin is combined wdth protein during 

 life, and its phosphoric groups may not be free to react with basic 

 dyes unless a fixative has acted.^*^ 



There is not one set of dyes that colours one sort of cytoplasmic 

 inclusion exclusively and another set that colours another. 

 Nevertheless, the dyes do not act quite unpredictably. Neutral 

 red has a strong tendency to colour vacuoles, and the phrase 

 'neutral red vacuoles' was at one time commonly used. There is, 

 however, no reason to suppose that all vacuoles colouring with 

 neutral red have important features in common, and it must be 

 remembered that this dye also colours many lipid globules. Most 

 vital dyes colour some kinds of lipid globules, while few of them 

 colour mitochondria. Dahlia, however, colours both lipid globules 

 and mitochondria, and Janus green B show^s some degree of 

 specificity for the latter (see p. 292). This specificity has, however, 

 been exaggerated, for it dyes strongly the oxyflavones that colour 

 the vacuoles of plant cells, and also certain plastids, though these 

 less strongly than mitochondria ^^^ ; it also dyes the external part 

 of the paranuclear bodies ('Golgi apparatus') in the primary 

 spermatocyte of the snail. *-^ Brilliant cresyl blue has a tendency to 

 dye the nucleolus, as the Lewises noted long ago in the course of 

 their w^ork on cell-culture. ^•^^ 



Certain cytoplasmic inclusions are coloured metachromatically 

 by particular vital dyes. A striking instance of this w^as reported 

 by Lauterborn -^^' ^^^ well over 60 years ago. He found that when 

 certain diatoms were stained vitally wdth methylene blue, the in- 

 clusions known as 'Biitschli's granules' were coloured reddish 

 violet. These granules contain or consist of the substance later 

 called 'volutin', that is to say, of macromolecular metaphosphates 

 (p. 246). It is probable that the metachromatic colouring attributed 

 by Lauterborn and other early workers to methylene blue was in 

 fact due to the highly metachromatic azures A and B, which always 

 form in old solutions of methylene blue (p. 268). At about the 

 same time Bolles Lee ^^^ noted vital metachromatic staining of the 

 Nehenkern of the primary spermatocytes of the snail with an old 



