58 ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS. 



(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 washing out is 

 complete, and must be stopped instantly. 



It is generally directed that absolute alcohol be taken for 

 washing out. This may be well in some cases, but in general 

 strong (95 per cent.) spirit 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, 

 the acid extracts the colour much more quickly from resting 

 nuclei than from kinetic nuclei, which is an advantage or a 

 disadvantage according to the end in view. 



The length of time necessary for washing out 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. 



In more than one of the methods presently to be described treatment 

 with chromic acid or with iodine forms part of the washing out process. 

 The rationale of this is somewhat obscure ; the most probable point of view 

 appears to be that the chromic acid acts as a mordant on the chromatin and 

 helps it to retain the stain. It is known on the one hand that chromic acid 

 precipitates safranin from its solutions, so that by admitting a special 

 affinity of chromic acid on the other hand for chromatin, and especially for 

 chromatin in the kinetic state, the explanation is hypothetically complete. 



The iodine in Bizzozero's (Gram's) process also appears to act as a fixative 

 of the colour. 



As a rough and ready guide to the beginner, it may be stated that wash- 

 ing out should be done with pure alcohol whenever it is desired to have rest- 

 ing nuclei stained as well as dividing nuclei ; the other processes serving 

 chiefly to differentiate " mitoses." 



98. Substitution. It was stated above that washing out is generally 

 done with alcohol. There exists another mode of washing out that is both 

 of practical importance and of great theoretical interest ; one anilin stain 

 may be 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 have been successfully carried out, 

 the nuclei stained with the first stain, the second forming a " contrast " 

 stain. It appeai-s from the interesting paper of RESEGOTTI in Zeit. f. wise. 



