ON STAINING WITH COAL-TAB COLOTJKS. 197 



is said that the solutions made with anilin had better be 

 employed only with preparations well fixed in chromo-aceto- 

 osmic acid, as the basic anil in oil may easilv attack chromatin 



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if not specially well fixed. 



Material fixed in chromo-osmic mixtures gives a sharper 

 and more .^elective stain than material fixed in sublimate or 

 the like. During- the staining the tissues become over stained, 

 that is charged with colour in an exc<->s<ive and diffuse 

 manner. The stain nmst therefore now be differentiated by 

 removal of the excess of colour. 



265. Differentiation. This is generally done with alcohol, 

 sometimes pure, 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 <-an. 

 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 Avater, 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 slowlv. The sections on the slide 



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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 colour, owing to the coming into view of the general 

 ground colour of the tissues, from which the stain has now 

 been removed. (Thus chrom-osmium- safranin sections turn 

 from an opaque red to a delicate purple.) At this point the 

 differentiation is complete, and the extraction of the colour 

 by the alcohol must be stopped instantly (see 267). 



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. 



The hydrochloric-acid-alcohol process had better only be 

 employed with tissues well fixed with " Flemming/' as with 

 tissues imperfectly fixed it may cause swellings. Further, 



