2^4 DYEING 



The fixatives that particularly favour acid dyes are trichloracetic 

 acid,*^^ picric acid, and chromium trioxide. Seki ^^^ w^ould add 

 potassium dichromate to these, but after this substance has been 

 allowed to act on the mammalian kidney, the cytoplasm of the 

 convoluted tubules is readily coloured by basic dyes.^^ The facts 

 suggest that trichloracetic acid, picric acid, and chromium trioxide 

 tend to block carboxyl groups and leave amino-groups free for 

 linkage with acid dyes. Seki, however, considers that basic proteins 

 are immobilized by the alkaloidal reagents and remain in the 

 tissues to react with acid dyes, while acidic proteins simply 

 dissolve away. Unfortunately we have not nearly so much know- 

 ledge about the reactions of these substances with proteins as 

 we have about the reactions of formaldehyde and mercuric 

 chloride. 



The effects of fixatives in blocking carboxyl or amino-groups 

 are reflected in changes in the iso-electric points of proteins. Such 

 changes are most readily observable with fibrin films, which can be 

 subjected to direct cataphoresis.^'^ The iso-electric point of un- 

 fixed fibrin is pH 6-0 ; denaturation by heating (i min. at ioo° C) 

 lowers this figure to 5-7; fixation by formaldehyde (10%, 16 hr.) to 

 5-2. Estimations of the shift of the iso-electric point of tissue- 

 constituents can be made by experiments with dyes used at 

 particular pHs.^^^' ^^^ It is found that various objects in the tissues 

 can be coloured by basic dyes at a lower pH after fixation by for- 

 maldehyde than after any other fixative. Ethanol has been found to 

 have little effect on the position of the iso-electric points of tissue- 

 constituents. 



The dyeing of chromatin is more complicated than that of the 

 cytoplasm. It is likely that fixatives generally split DNA from 

 protein, and the colouring is then mainly that of DNA by the 

 basic dye.^^^ A virtue of acetic acid is that it leaves chromatin 

 scarcely colourable by acid dyes, so that basic dyes are not masked. 

 Many other fixatives allow strong coloration of chromatin by basic 

 dyes: for instance, mercuric chloride, formaldehyde, ethanol, and 

 potassium dichromate. The two latter, however, do not coagulate 

 nucleoproteins, and there is therefore no immobilization of 

 chromatin in its original sites. When it is contained in the nucleus, 

 however, it is prevented from escape by the nuclear membrane, and 

 it distributes itself almost at random within the nucleus. This is 

 particularly obvious when one examines the first meiotic prophase 

 in a gonad fixed in ethanol or potassium dichromate. No one would 



