160 COMBINATION STAINS. 



As regards sections stained by the indirect or Flemming's 

 method, considerable latitude is allowable in the manipula- 

 tions. Some persons stain first in the secondary stain (eosin, 

 for instance), then wash and stain in the primary stain (by 

 "primary" stain I constantly mean the nuclear stain), and 

 wash out until the colour of the secondary stain reappears. 



Another method, which will, I think, frequently be found 

 preferable as allowing a stronger primary stain, is to stain 

 first with the primary, say gentian violet, and then with the 

 secondary, say eosin. Care must be taken not to wash out 

 the first stain as omnpletely as if you were going to mount it 

 at once, else the operations required by the second stain may 

 result in entirely removing the colour of the first. 



Foremost amongst these processes must be mentioned 

 FLEMMING'S Safranin, Gentian, and Orange Stain (Arch. f. mile. 

 Anat. f xxxvii, 1891, p. 249; ibid., p. 685; Zeii.f. wiss. Mik., 

 viii, 2, 1891, p. 223, and viii, 3, p. 343). This is a substitu- 

 tion method. You stain (for as much as two or three days if 

 you want a very powerful stain) in strong alcoholic safranin 

 solution diluted with anilin water ( 101) ; rinse in distilled 

 water ; wash out in absolute alcohol, containing at most O'l 

 per cent, of hydrochloric acid, until hardly any more colour 

 comes away ; stain for one to three hours in gentian (either 

 a simple very strong aqueous solution, or, if you prefer, you 

 may stain by the method of Gram, 102) ; wash for a short 

 time in distilled water ; treat with concentrated, or at least 

 fairly strong, aqueous solution of orange, which in virtue of 

 its acid properties washes out most of the gentian. After at 

 most a few minutes, whilst pale violet clouds are still being 

 given off from the sections on agitation, bring them into abso- 

 lute alcohol until hardly any more colour comes away, clear 

 in clove or bergamot oil, and mount in damar or balsam 

 before the last pale clouds of colour have ceased to come 

 away (the orange should be the orange G, introduced into 

 commerce by Meister, Lucius, and Bruning, of Hochst, and 

 also prepared by the Actiengesellschaft fur Anilinfabrication, 

 Berlin ; it may be obtained from GRUBLER, 94). 



This is not a triple stain in the sense of giving three 

 different colours in the result ; the orange does not act as a 

 separate stain, but as an agent for the differentiation of the 

 gentian stain. Chromatin and nucleoli are purple-red ; achro- 



