226 DYEING 



blue. It is characteristic of all trapping agents that they precipitate 

 certain dyes from aqueous solutions, and that the precipitated 

 material has a low solubility in alcohol. Thus crystal violet is 

 freely soluble in 95 % alcohol, but the crystal violet/potassium tri- 

 iodide precipitate will only dissolve at concentrations up to 0-07%. 

 The crystal violet/potassium permanganate precipitate is even less 

 soluble (up to 0-02%). All basic dyes that have been tested are 

 precipitated by iodine, but a few of the precipitates are fairly 

 soluble in 95% alcohol. Thus the neutral red precipitate dissolves 

 at 0*43% and the rhodamine B at o-6i %. These dyes would not be 

 selected for work with trapping agents. Very few acid dyes are 

 precipitated by iodine. Acid fuchsine and aniline blue WS are 

 exceptional in this respect. ^^' *" 



Potassium permanganate traps crystal violet so effectively that it 

 cannot take the place of iodine in the Gram technique, for it holds 

 the dye even in Gram-negative bacteria. ^^ 



The way in which trapping agents work has not been fully 

 established. Two possibilities present themselves. On one hand it 

 may be that the basic dye attaches itself as usual to acidic objects 

 and remains there subsequently in the presence of an extracting 

 agent (ethanol) simply because iodine is also present, which in- 

 stantly precipitates any dye that begins to be extracted. On the 

 other hand it is possible that an object/dye/iodine complex is 

 formed, which is not easily split by alcohol. 



If the first possibility is true, the continued presence of iodine 

 all round the object is obviously necessary. The Gram method 

 depends upon the fact that iodine can enter Gram-positive 

 bacteria in the form of potassium tri-iodide, but its outward 

 diffusion in the form of iodine/ethanol (which is also brown) may 

 be hindered by factors that did not prevent its entrance. It may 

 be that Gram-positive bacteria have an external part, perhaps a 

 special membrane or cell- wall, ^^ that hinders the escape of 

 iodine/ethanol. The density and permeability of the Gram- 

 positive substance itself may also affect the escape of iodine. If 

 iodine can escape, the dye will be extracted by alcohol. 



On this view a bacterium could be Gram-negative because the 

 dye could not enter it, or because there was little or no acidic 

 material in it to hold a basic dye, or again because there was no 

 special cell- wall or other feature having the property of hindering 

 the escape of iodine. It is noteworthy in this connexion that 

 if Gram-positive bacteria be crushed, they appear Gram- 



