40 ESSENTIALS OF BACTERIOLOGY. 



To Stain Spores. Since spores have a very firm capsule, 

 which tends to keep out all external agents, a very intensive 

 stain is required to penetrate them, but once this object attained 

 it is equally as difficult to decolorize them. 



A cover-glass prepared in the usual way, i. e., drying and 

 passing the specimen through the flame three times, is placed in 

 a watch-crystal containing Ziehl's carbol-f'uchsin solution, and 

 the same placed upon a rack over a Bunsen burner, where it is 

 kept at boiling-point for one hour, careful to supply fresh solution 

 at short intervals lest it dry up. 



The bacilli are now decolorized in alcohol, containing per 

 cent, hydrochloric acid. A contrast color, preferably methylin 

 blue, is added for a few minutes. 



The spores will appear as little red beads in the blue bacteria, 

 and loose ones lying about. 



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, 2 seconds ; rinsed in water. 



III. Methylin blue contrast. 



Alex. Klein recommends the following spore method : mix a 

 little of the culture (potato) with 3 drops of physiologic salt 

 solution, and heat gently with an equal quantity of carbol- 

 fuchsin for a period of 6 minutes. Spread then on cover-glasses, 

 dry in the air, and fix by passing three times through Bunsen 

 burner flame. Decolorize in 1 per cent, sulphuric acid for 1 to 

 2 seconds ; contrast in weak methylin blue. 



BowhilVs Orcein Stain. 



Sat. alcoholic solution of orcein . . .15 c.c. 

 20 per cent, aqueous sol. 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 10 minutes. Wash in water. Dry and 

 mount in balsam. 



Five per cent, chromic acid applied for 15 minutes has been 

 recommended in staining spores. This is followed by the 

 carbol-fuchsin stain as above. 



