236 DYEING 



purpose it will suffice to concentrate on collagen and cytoplasm. 

 The dyes that colour the cytoplasm from these pairs are mostly the 

 ordinary background dyes used for contrast with the basic dyes 

 that colour chromatin. The most typical of these are eosin 

 (xanthene), orange G (azo), ponceau 2R (azo), and especially 

 picric acid (nitro). It is interesting to notice that most of the dyes 

 used in microtechnique for colouring the background are levelling 

 dyes. 



The most typical of the dyes for collagen are aniline blue WS 

 and the closely related methyl blue (triarylmethane), induline WS 

 and nigrosine W (azine), diamine blue 2B and naphthol black B 

 (azo), and indigo-carmine (indigo dye). The striking fact about 

 this apparently random set of dyes is that none of them belongs to 

 the 'levelling' group. Methyl blue, for instance, can be used for 

 cotton, and is indeed sometimes called 'cotton blue'; diamine 

 blue 2B is a direct dye for cotton; and others in this group, to a 

 greater or lesser extent, have the characters of milling dyes. 



In brief, then, the feeble levelling dyes colour the cytoplasm, the 

 vigorous milling and cotton dyes colour collagen. Why? 



The first clue was obtained in the twenties by a Frenchman, 

 Collin, ^2^' ^^^ who made a special study of Mann's methyl blue/ 

 eosin. ^^^' ^^^ He dissolved gelatine at various concentrations in 

 warm water, dipped microscopical glass slides in these solutions, 

 dried the films thus produced, and fixed them in a mixture of 

 formaldehyde and alcohol. After washing and again drying them 

 he put them in Mann's mixture. The films made from concen- 

 trated solutions of gelatine took up the red colour of eosin, those 

 from weak solutions the blue of methyl blue. 



These results suggested that methyl blue was not able to enter 

 concentrated gelatine, but that wherever it could enter and com- 

 pete with the eosin, it dominated the latter. In other words, the 

 blue dye was more vigorous in action, but penetrated with 

 greater difficulty than the red. 



Collin now showed that eosin penetrated much more rapidly 

 than methyl blue into gelatine gel contained in a test-tube, and 

 also that if Mann's mixture was put in a collodion sack, and the 

 sack in water, the water was coloured by eosin before the methyl 

 blue escaped. A solution of methyl blue, filtered, contains particles 

 that are visible under the microscope: a solution of eosin does 

 not. Various objects (blotting-paper, animal charcoal, etc.) take 

 up much more methyl blue than eosin from solutions at the 



