COAL-TAR CHROMATIN STAINS. 209 



agitating the aqueous solution with chloroform. FISCHER (Fixirung, 

 Fdrbung, u. Bau d. Protoplasmus, p. 89) shakes up the solution in a burette 

 with a little amyl alcohol, which quickly becomes violet and collects at the 

 top, whence it may be decanted off and the operation repeated. 



Methyl green is extremely sensitive to the action of alkalies. 

 It is therefore important to use it only in acidified solutions, 

 and to use only acid, or at least perfectly neutral fluids for 

 washing and mounting. 



This is an extremely important histological reagent. Its 

 chief use is as a chromatin stain for fresh, unfixed tissues. 

 For this purpose it should be used in the form of a strong 

 aqueous solution containing a little acetic acid (about 1 per 

 cent, in general). Tfie solutions must always be acid. (If 

 the tissues have been- previously fixed with acetic acid you 

 will not get a chromatin stain. The same applies to fixation 

 with acetic acid sublimate : whilst pure sublimate will allow 

 of a chromatin stain (BUKCKHAKDT, La Cellule, xii, 1897, 

 p. 364). You may wash out with water (best Aidulated) 

 and mount in some acid aqueous medium containing a little 

 of the methyl green in solution. The mounting medium, if 

 aqueous, must be acidulated. 



Employed in this way, with fresh, unfixed tissues, methyl 

 green is a pure chromatin stain, in the sense of being a 

 precise colour reagent for chromatin. For in the nucleus it 

 stains nothing but chromosomes, or chromatin elements : it 

 does not stain plasmatic nucleoli, nor caryoplasm, nor achro- 

 matic filaments. Outside the nucleus it stains some kinds of 

 cytoplasm and some kinds of formed material, especially 

 glandular secretions (silk, for instance, and mucin). The 

 chromatin elements are invariably stained of a bright green 

 (with the exception of the nuclein of the head of some 

 spermatozoa), whilst extra-nuclear structures are in general 

 stained in tones of blue or violet. But this metachromatic 

 reaction is probably due to the methyl-violet impurity, and 

 is not obtained with a chemically pure methyl green. 



Besides being a perfectly precise test for chromatin in the 

 fresh nucleus, methyl green has other advantages. Staining 

 is instantaneous ; overstaining never occurs. The solution 

 is very penetrating, kills cells instantly without swelling or 

 other change of form, and preserves their forms for at least 

 some hours, so that it may be considered as a delicate fixa- 



14 



