42 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. Carbol-fuchsin 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 carbol- 

 fuchsin 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. 



Bowhill's Orcein Stain. 



Saturated alcoholic solution of orcein . . 15 c.c. 



20 per cent, aqueous solution tannin ... TO 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 carbol-fuchsin 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 Kuhne mixes the alcohol 

 with the stain, so that while the section is being anhydrated, it 

 is constantly supplied with fresh dye. 



