128 COMBINATION STAINS. 



As regards sections stained by the indirect or Flemming's 

 method, considerable latitude is allowable in the manipulations. 

 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 completely 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. 



Lists of some colours that give good results as primary and 

 as secondary stains are given in 98. Besides the combina- 

 tions there recommended I would particularly recommend 

 that of Gentian Violet and Eosin. Stain and wash out (not 

 too far) by Bizzozero's method ( 102), and then stain for a 

 few seconds, or as much as two to five minutes, in fairly strong 

 aqueous solution of eosin, dehydrate rapidly with alcohol, 

 clear and mount. 



Attention should be paid to the decolorising action of the 

 secondary stain on the primary (see 98). This reaction may 

 be utilised, as proposed by BAUMGAETEN (infra 246). 

 RESEGOTTI (Zeit.f. wiss. Mik., v, 3, 1888, p. 323) recommends 

 staining for five minutes (this is for tissues fixed by alcohol) 

 in strong solution of Methyl Violet or Dahlia, then washing out 

 for one or two minutes in very weak solution of (alcoholic) 

 Eosin or of Saurefuchsm in alcohol, then dehydrating and 

 clearing. 



In the following paragraphs are given some other methods 

 of proved utility. 



246. Baumgarten's Fuchsin and Methylen Blue (Zeit.f. wiss. 

 Mik., i, 1884, p. 415). Stain sections (of chromic objects) for 

 twenty-four hours in a stain made by adding 8 to 10 drops of 

 concentrated alcoholic solution of fuchsiu to a watch-glassful 

 of water. Rinse with alcohol, and stain for four or five 

 minutes in concentrated aqueous solution of methylen blue, 

 wash out with alcohol for five to ten minutes, and clear with 

 clove oil. Nuclei red, tissues blue, the fuchsin having been 



