THE INDIRECT ATTACHMENT OF DYES TO TISSUES 225 



Only eight are known, however, that give a satisfactory 'true 

 Gram-positive' reaction, and these are all triarylmethanes.*^' ^'^ It 

 w^as formerly held that pararosanilines were suitable, while 

 rosanilines were not.^^^ It may be recollected that rosaniline has a 

 methyl group attached to one of the three ar}d rings, while 

 pararosaniline lacks this (p. 159). Dahlia, however, is one of the 

 eight dyes that give a satisfactory 'true Gram-positive': yet this is 

 one of the rosanilines, for it possesses the methyl group. 



Gram-positive and Gram-negative bacteria can be distinguished 

 by the use of crystal violet alone, without any trapping agent. *^ 

 The differentiation in alcohol is so difficult, however, that the 

 technique is not suitable for routine use in the bacteriological 

 laboratory. It seems that iodine is used because it is extremely 

 convenient rather than because it is theoretically necessary. 



There is very strong evidence that the substance that retains 

 crystal violet in Gram-positive bacteria is a ribonucleopro- 

 tein.^^^' 226, 48 j^ j^^y \^Q mentioned, however, that this is not 

 undisputed. ^26 j^ jg certain that a positive Gram reaction does not 

 always denote the presence of RXA. Indeed, one would not expect 

 this to be so, for cr}^stal violet will behave like other basic dyes and 

 is likely to be held by iodine wherever its affinity for acidic objects 

 has caused it to be present in particularly large amount. In the 

 spermatozoon of Ascaris, for instance, there is a large cytoplasmic 

 inclusion, the 'refringent cone', which is an object consisting of 

 highly acidic (basiphil) proteins. This naturally takes up a lot of 

 crystal violet and is strongly Gram-positive. ^^2 it contains no RNA 

 whatever. 



The use of iodine to trap gentian violet in chromatin and thus 

 allow slower differentiation in alcohol was introduced by Her- 

 mann, 228 whose technique involved the use of another dye as well. 

 Gentian and crystal violets, trapped by iodine, are much used in 

 modern chromosome studies.^^"' 247,278,39 ^j^g method is valuable, 

 for the cytoplasm is of glassy transparency, while the delicate 

 chromosomal threads of early meiosis retain the dye. 



Iodine is the most familiar trapping agent. It is seldom used in 

 its blue, molecular form, but is nearly always dissolved in aqueous 

 potassium iodide solution and thus presents itself as brown potas- 

 sium tri-iodide, KI3. Various other trapping agents are available, 

 such as bromine, mercuric iodide, mercuric chloride, potassium 

 permanganate, and picric acid.^^^' •*" In vital studies, as we shall 

 see (p. 294), ammonium molybdate is used to trap methylene 

 p 



