136 STAINING 



their physical state is more or less altered. Alcohol is one of these 

 agents. Other fixing fluids, of which those containing chromic 

 acid are representatives, produce what seem to be compounds of 

 cell proteins with the reagent. In this latter case, we have what is 

 known as a " mordant " present. 



Mordants are usually understood to be agents which form 

 insoluble compounds with dyes and in this way cause their 

 fixation in places from which otherwise they might be washed 

 out by the subsequent treatment with dehydrating agents, etc. 

 Such a substance may clearly be either already present in the 

 fixed preparation when the dye is added, or it may be added 

 together with or subsequently to the staining agent. It will 

 readily be understood that the production of a hardly soluble dye 

 salt renders solution in water more difficult, but this alone is not 

 enough. The compound must also be firmly attached to the 

 surface. 



The insoluble compounds between a dye and a mordant are 

 known as " lakes," But the chemical nature of these substances 

 is by no means clear — especially when they are produced in situ 

 in stained tissues. In fact, they do not behave as simple com- 

 pounds of the dye and the mordant. They resist the action of 

 strong acids and bases in moderately strong solutions, so that the 

 stains obtained by this method are characterised by durability 

 and " fastness." 



On the whole, we must conclude that there are factors still 

 unknown, but possibly related to the electrical properties of the 

 surface, which play an important part in these reactions with 

 mordants. Neither a simple chemical theory nor one purely 

 physical accounts for all the phenomena. 



A further mention may here be made of the " progressive " 

 and " regressive " methods. A preparation may be placed in a 

 very dilute solution of a dye and the action stopped when the 

 elements with the greatest " affinity " for the dye have taken it 

 up. The staining of nuclei with dilute alum-hasmatoxylin may 

 be mentioned. If the action is prolonged, various other con- 

 stituents of the cell, besides the nucleus, take on the colour. As 

 a rule, no great differentiation is obtained by this method. Better 

 results are given by the " regressive " method, in which a general 

 overstaining is followed by a partial decoloration, in which 

 certain elements retain the stain, owing to special chemical or 

 physical properties, after it has been removed from the rest. 

 Thus safranin stains the whole section a deep red colour ; 

 but alcohol removes the stain from all but the chromatin 

 and the nucleoli. This action of alcohol may be explained, as 

 already iJointed out, by its effect on the magnitude of the electric 

 charge, since its dielectric constant is lower than that of water. 

 Other differentiating agents are also used. Iron-alum, in the 



