60 ESSENTIALS OF BACTERIOLOGY 



The spores will appear as little red beads in the blue-stained 

 bacteria, and loose spores lying about outside the cell- wall. 



Spore Stain (Modified). I. Carbolfuchsin on cover-glass 

 and heated in the flame to boiling-point 20 to 30 times. 



II. 25 per cent, sulphuric acid, two seconds; rinsed in 

 water. 



III. Methylene-blue contrast. 



Alex. Klein recommends the following spore method: mix a 

 little of the culture (potato) with three drops of physiologic 

 salt solution, and heat gently with an equal quantity of 

 carbolfuchsin for a period of six minutes. Spread then on 

 cover-glasses, dry in the air, and fix by passing three times 

 through Bunsen-burner flame. Decolorize in i per cent, 

 sulphuric acid for one to two seconds; contrast in weak 

 methylene-blue. 



BowhilFs Orcein Stain 



Saturated alcoholic solution of orcein . 15 c.c. 



20 per cent, aqueous solution tannin . 10 c.c. % 



Distilled water 30 c.c. M. 



Filter. 



Use orcein solution in watch-glass, float cover-glass in it, 

 and heat gently, not boil, for ten minutes. Wash in water. 

 Dry and mount in balsam. 



Five per cent, chromium trioxid applied for fifteen minutes 

 has been recommended in staining spores. This is followed 

 by the carbolfuchsin stain as above. 



Sporogenic bodies stain quite readily, and in order to distin- 

 guish them from spores Ernst uses alkaline methylene-blue, 

 slightly warmed. Then rinse in water. Contrast with cold 

 Bismarck-brown. The spores are colored bright blue, the 

 spore granules a dirty blue, being mixed with the brown, 

 which colors also the bacteria. 



Kiihne's Method. In sections the alcohol used sometimes 

 decolorizes too much. To obviate this Kiihne mixes the alco- 



