STAINING, PRACTICAL AND THEORETICAL 



overwhelming number of free nigrosin molecules whose colour 

 has not been affected. It would seem that in uniting with nigrosin 

 the eosin becomes detached and carried away from the cytoplasm. 

 But in this case, the eosin is not replaced by the other acid dye 

 (nigrosin) for some reason, not understood, but probably due to 

 physical phenomena. The number of molecules of nigrosin 

 reduced in colour by union with the eosin would, as already stated, 

 be infinitesimal compared with the large excess of undecolorized 

 nigrosin molecules. Therefore no reduction in the colour of the 

 nigrosin as a whole would be observable. 



Rosenbaum (1949) states that while it is generally agreed that 

 any staining process involves both physical and chemical factors, 

 the physical phenomena which occur during negative staining 

 with nigrosin are of particular interest. As Rosenbaum remarks in 

 his paper which deals with the negative staining of protozoa with 

 water soluble nigrosin, negative staining consists essentially of 

 staining the background of the field of observation, leaving the 

 actual specimen unstained. He also states that all workers have 

 reported water-soluble nigrosin as a strong decolorizing agent, 

 and that in his opinion the dye depends upon colloidal reactions 

 in its staining effects. Along these lines, Burri (1909) mentions 

 Indian ink as ideally suited for negative-staining. Indian ink, 

 Rosenbaum states, produces an opaque background film of suitable 

 thickness for the process. Nigrosin, on the other hand, has the 

 ability to disperse more freely in a liquid phase, and hence the 

 resulting suspension is more fluid than Indian ink. The same 

 author (Rosenbaum, 1949) states that the results of earlier work 

 carried out by him on the effects of certain dye suspensions on 

 Pelomyxa suggest that homogeneous particles of water-soluble 

 nigrosin, form, in their dispersed phase, close contact with each 

 other on body surfaces and in solution so as to produce a high 

 surface tension of great capillarity. 



This also, I would suggest, is probably a contributory factor in 

 decolorizing action of water-soluble nigrosin. 



Notes on 

 THE pH OF DYES IN AQUEOUS SOLUTION 



It should be mentioned here that a true acid dye, as used in 

 histology, is a metallic salt (but sometimes an ammonium salt) 



16 



