88 APPLIED BACTERIOLOGY 



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 unstaiued 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 stained 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 

 with Loffler's methylene blue for three minutes, washed in 

 water, blotted, dried and mounted. Examined with a 

 yV inch oil immersion lens, the bacilli will be found to be 

 stained blue and the spores red. 



This method gives very satisfactory and pretty prepara- 

 tions. The spores of Bacillus megatherium and Bacillus 

 Mamentosus are more easily stained than those of Bacillus 

 anthracis or the hay bacillus {Bacillus subtilis). 



Fiocca's Method. — This method is also very successful and 

 rapid. About 1 c.c. of a saturated alcoholic solution of 



