METHODS OF CULTURE. 39 



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-tuchsin 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 methyliu 

 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. 



Flagella Stain, with Loffler^s Mordant. I. A few drops of the 

 mordant (No. viii.) are placed upon the spread cover-glass and 

 heated until it steams. 



II. Washed with water until the cover-glass looks almost clean, 

 using a small piece of filter paper to rub off the crusts which have 

 gathered around the edges. 



III. Aniline water fuchsin (neutral) held in flame about 1 

 minutes. 



IV. Wash in water. 



If the stain is properly made, the microbes are deeply colored 

 and the flagella seen as little dark lines attached to them. 



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

 guish them from spores Ernst uses alkaline methylin blue, slightly 

 warmed. 



Then rinse in water. 



Contrast with cold bismark-brown. 



The spores are colored bright blue, the spore granules a dirty 

 blue, being mixed with the brown, which colors also the bacteria. 



Kuhne's Method. In sections,, the alcohol used sometimes de- 

 colorizes too much. To obviate this Kiihne mixes the alcohol 

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

 is constantly supplied with fresh dye. 



Weigert uses aniline oil to dehydrate instead of alcohol, and 

 here, too, it can be used mixed with the dye. 



