CHAPTER XIV. 163 



it is frequently well to leave the sections twelve to twenty-four hours 

 in the fluid. Up to a certain point the more the tissues are stained 

 the better do they resist the washing-out process, which is an 

 advantage. Some workers, indeed, prefer weak solutions ; so 

 HEIDENHAIN, EncycL mik. Technik, i, pp. 433, 434 ; but the nature 

 of the fixing agent should be taken into account. 



Material fixed in chromic or chromo-osmic mixtures gives a 

 sharper and more selective stain than material fixed in sublimate or 

 the like. In fact, to ensure the best results, only material fixed in 

 chromic mixtures (or Hermann's fluid) should be employed. 



During the staining the tissues become overstained, that is, charged 

 with colour in an excessive and diffuse manner. The stain must 

 now be differentiated by removal of the excess of colour. 



281. Differentiation. This is generally done with alcohol, some- 

 times neutral, sometimes acidulated (with HC1). 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 chrom-osmium objects) have changed 

 colour, owing to the coming into view of the general ground colour 

 of the tissues. (Thus chrom-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 differen- 

 tiation. This may be well in some cases, but in general 95 per cent, 

 is found to answer perfectly well. HEIDENHAIN (EncycL, 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. 



112 



