182 NUCLEAR STAINS, COAL TAR 



stain must now be differentiated by removal of the excess of 

 colour. 



367. Differentiation. This is generally done with alcohol, 

 sometimes neutral, sometimes acidulated (with HCl). The stained 

 sections, if loose (celloidin sections), are brought into a watch- 

 glassful of alcohol ; if mounted in series on a slide, they are 

 brought into a tube of alcohol (differentiation can be done by 

 simply pouring alcohol on to the slide, but it is better to use a 

 tube or other bath). It is in either case well to just rinse the 

 sections in water, or even to wash them well in it, before bringing 

 them into alcohol. 



The sections in the watch-glass are seen to give up their colour 

 to the alcohol in clouds, which are at first very rapidly formed, 

 afterwards more slowly. The sections on the slide are seen, if 

 the slide be gently lifted above the surface of the alcohol, to be 

 giving off their colour in the shape of rivers running down the glass. 

 In a short time the formation of the clouds or of the rivers is seen 

 to be on the point of ceasing ; the sections have become pale and 

 somewhat transparent, and (in the case of chrome-osmium objects) 

 have changed colour, owing to the coming into view of the general 

 ground colour of the tissues. (Thus clirome-osmium-safranin 

 sections turn from an opaque red to a delicate purple.) At this 

 point the differentiation is complete, or nearly so. 



It is generally directed that absolute alcohol be taken for 

 differentiation. This may be well in some cases, but in general 

 95 per cent, is found to answer perfectly well. Heidenhain 

 {Enzyk., i, p. 434) takes methyl alcohol. 



The hydrochloric-acid-alcohol extracts the colour much more 

 quickly from resting nuclei than from kinetic nuclei. Therefore, 

 washing out should be done with neutral alcohol whenever it is 

 desired to have resting nuclei stained as well as dividing nuclei ; 

 the acid process serving chiefly to differentiate karyokinetic figures. 



The proportion of HCl with which the alcohol should be acidified 

 for the acid process should be about 1 : 1000 or less ; seldom 

 more. 



The length of time necessary for differentiating to the precise 

 degree required varies considerably with the nature of the tissues 

 and the details of the process employed ; all that can be said is 

 that it generally lies between thirty seconds and two minutes. 

 The acid process is vastly more rapid than the neutral process, 

 and therefore of course more risky. 



There exists also a method of differentiation known as substitution — 

 one stain being made to wash out another. Thus methylen blue and 

 gentian violet are discharged from tissues by aqueous solution of vesuvin 

 or of eosin ; fuehsin is discharged from tissues by aqueous solution of 

 methylen blue. The second stain " substitutes " itself for the first in 

 the general " ground '" of the tissues, leaving, if the operation has been 



