174 CHAPTER XIV. 



281. 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 i-apidly 

 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 trans- 

 parent, and (in the case of chrom-osmium objects) have 

 changed coloiir, owing to the coming into view of the general 

 ground colour of the tissues. (Thus ohrom-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 pei-fectly well. 

 Heidenhain [Encycl., i, p. 434) takes methyl alcohol. 



The hydrochloric-acid-alcohol extracts the colour much 

 more quickly from resting nuclei than from Tcinetic 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 liaryohlnetic 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 riskJ^ 



