NUULHAR STAINS WITH COAL-TAB DYES. 175 



There exists also a method of differentiation known as siibstitution — 

 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 ; f uchsin 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 Besegotti 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 Besegotti 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 differentiationj the extraction of 

 the colour may be stopped by putting the sections into 

 water ; but the general practice is to clear and mount theui 

 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 aniline than clove oil. 



Series of sections on slides are conveniently cleared by 

 pouring the clearing agent over them. 



After clearing you may either mount at once in damar or 

 balsam ; or, stop the extraction of the colour, if clove oil 

 have been used, by putting the sections into some medium 



