STAINING OF SPORES 89 



Staining of Spores. — The spores of microbes differ from 

 the fully-formed organisms in the resisting power they offer 

 to staining solutions. When ordinary cover-glass prepara- 

 tions, stained in the usual way, are made of some organ- 

 isms — say, for instance, of Bacillus anthracis or Bacillus 

 megatherium — bright unstained spots are sometimes seen, 

 which may be isolated or in the middle or ends of the 

 organisms. These are the spores which have resisted the 

 colouring matter of the stain. All unstained spots in pre- 

 parations are not necessarily spores, as many causes may 

 give rise to this irregular appearance, among which may be 

 mentioned faulty staining due to air-bubbles, the use of old 

 staining solutions, or in the case of old cultures the organ- 

 isms may have become degenerate and broken down ; these 

 and many other causes may give rise to unstained spots 

 which may be mistaken for spores. 



Heat Method. — If an ordinary cover-glass preparation is 

 passed through the flame about twelve times instead of 

 three, as is usual in ' fixing,' stained for a few minutes 

 with warm Ehrlich's gentian violet or Ziehl's fuchsine 

 solution, and then well washed in water, the spores will 

 be found to be deeply stained, whereas the bacilli will 

 be found to be only faintly staiaed or colourless. The 

 heating destroys the power of the organisms to take up 

 the stain, thus leaving only the spores stained. 



Neisser's Method. — The cover-glasses, prepared in the 

 usual way, are stained with warm carbol-fuchsine solution 

 for about thirty minutes. For this purpose it is best to 

 float the cover-glasses on the surface of the stain contained 

 in a small dish on a sand-bath, which is kept warm with 

 a very small flame. The cover-glass is removed, washed 

 in water, and then decolourised for a few seconds in a 3 per 

 cent, alcoholic solution of hydrochloric acid. The cover- 

 glass is now well washed in water, and counter-stained 



