48 MANUAL OF BACTERIOLOGY 



that the solution may not be completely saturated. The purpose of the above 

 procedures is to modify the methylene-blue so that other staining elements are 

 developed in it (poly chromism) . The modified methylene-blue solution is 

 then combined with eosin. For further details see Wright. Journal of Medical 

 Research. Vol. VII., 1902. 



Beside the various modifications of the Romano wsky stain given above, a 

 number have been recommended, all of them having this in common that the 

 nuclear stain depends upon the modification of methylene-blue due to the 

 action of dilute alkali. Giemsa has put on the market a ready prepared stain 

 which is essentially a modified Romanowsky. The trade name for the prep- 

 aration is "Asur I. (pur.)". "Asur II" is a mixture of Asur I with equal 

 parts of methylene-blue. 



Staining of Spores. The method is applicable to cover- 

 glass preparations which may be prepared in the usual way 

 from material containing spores. 



(a) After drying the smear on the cover-glass, and fixation 

 with heat by passing through the flame three times, use aniline- 

 water fuchsin or carbol-fuchsin as a stain. 



(b) Heat until the preparation begins to boil; remove for a 

 minute; heat again, and again remove; repeat this process six 

 times. 



(c) Wash in dilute alcohol (Novy) or in a weak solution of 

 "acetic or hydrochloric acid for a few seconds to a minute. 

 Some spores are quickly decolorized by i per cent, acetic acid; 

 others may keep the stain when subjected to 3 per cent, 

 hydrochloric acid alcohol for a minute. 



(d) Wash in water. 



(e) Stain with watery solution of methylene-blue half a 

 minute. 



(/) Wash. 



fe) Dry. 



(h) Balsam. 



The spores are intensely stained by the fuchsin. The stain 

 is removed from everything except the spores by the acid. 

 The methylene-blue solution stains the bodies of the bacteria, 

 the spores remaining brilliant red. There are various other 



