164 NUCLEAR STAINS WITH COAL-TAR DYES. 



The proportion of HC1 with which the alcohol should be acidi- 

 fied 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 ; fuchsin 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 

 successfully carried out, the nuclei stained with the first stain, the 

 second forming a " contrast " stain. In the paper of RESEGOTTI in Zeit. 

 wiss. Mik., v, 1888, p. 320, it is stated as a very general rule that colours 

 that do not give a nuclear stain by the regressive method will wash out 

 those that do. But RESEGOTTI used the second colour in alcoholic 

 solution ; so that it remains uncertain how far the differentiation should 

 be attributed to the second colour itself, and how far to the alcohol 

 used, as a vehicle. The same remark applies to BENDA'S Safranin-and- 

 Lichtgriin process. 



282. Clearing. After due differentiation, the extraction of the 

 colour may be stopped by putting the sections into water ; but the 

 general practice is to clear and mount them at once. 



You may clear with clove oil or anilin, which will extract some 

 more colour from the tissues. Or you may clear with an agent that 

 does not attack the stain (cedar oil, bergamot oil, xylol, toluol, etc. ; 

 see the chapter on Clearing Agents). If you have used neutral 

 alcohol for washing out, you had perhaps better clear with clove oil, 

 as neutral alcohol does not always, if the staining have been very 

 prolonged, extract the colour perfectly from extra-nuclear parts. 

 But if you have not stained very long, and if you have used acidulated 

 alcohol for washing out, clove oil is not necessary, and it may be 

 better not to use it, as it somewhat impairs the brilliancy of the 

 stain. A special property of clove oil is that it helps to differentiate 

 karyokinetic figures, as it decolours resting nuclei more rapidly than 

 those in division. 



Some colours are much more sensitive to the action of clove oil 

 than others ; and much depends on the quality of this much- 

 adulterated essence. New clove oil extracts the colour more quickly 

 than old, and anilin than clove oil. 



