NUCLEAR STAINS, COAL TAR 183 



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

 second forming a " contrast " stain. In the paper of Kkhegotii 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. 



368. 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, zvhich zvill 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 pro- 

 perty 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. 



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 that does not affect 

 the stain (xylol, cedar oil, etc.). Chloroform should be avoided, 

 either as a clearer or as the menstruum for the mounting medium. 



369. General Results. The results depend in great measure on 

 the previous treatment of the tissues. If you have given them a 

 prolonged fixation in Flemming's strong chromo-aceto-osmic 

 mixture, and have differentiated after staining with acid alcohol 

 and cleared with clove oil, you will get, with some special excep- 

 tions, nothing stained but nucleoli and the chromatin of dividing 

 nuclei, that of resting nuclei remaining unstained. If you have 

 given a lighter fixation, with Flemming's weak mixture or some 

 other fixing agent not specially inimical to staining, and have 

 differentiated after staining with neutral alcohol, you will get the 

 chromatin of resting nuclei stained as well. Either process may 



