104 VETERINARY BACTERIOLOGY 



directly upon the glass microscopic slides. The procedure is 

 practically identical with that detailed above for cover-glass 

 preparations except that the immersion oil may be placed directly 

 upon the stained film and no cover-glass used. 



Spore Stain. Bacterial spores stain with difficulty, but once 

 stained do not yield up the stain readily. Either one of the 

 following methods will be found to give good results : 



Hansen Method. 1. Prepare a film, fix, and stain with steam- 

 ing hot carbol-fuchsin for five minutes. 



2. Decolorize with 5 per cent, acetic acid until the film is a light 

 pink, and wash in water. 



3. Stain three minutes with Loffler's methylene-blue. 



4. Examine. 



M oiler's Method. 1. Prepare films and fix in chloroform for 

 two minutes. 



2. Dry in air. 



3. Cover with 5 per cent, solution of chromic acid for two 

 minutes. 



4. Wash in water. 



5. Stain with hot carbol-fuchsin five minutes. 



6. Decolorize with 1 per cent, sulphuric acid twenty-five to 

 thirty seconds. 



7. Wash and counterstain with methylene-blue ten to fifteen 

 seconds. 



8. Examine. 



By either method the spores appear red and the cell body blue. 



Stain for Acid-fast (Acid-proof) Organisms. Certain bacteria 

 are stained with difficulty, but when once stained, they resist 

 decolorization with acids. The most important of these organisms 

 is Bacillus tuberculosis. 



Acid Alcohol Method. 1. Prepare film, fix in flame, and stain 

 with hot carbol-fuchsin for two minutes. 



2. Wash in 2 per cent, hydrochloric acid in 95 per cent, alcohol 

 until there is no color visible in the thinner portions of the film. 



3. Wash in water and stain with methylene-blue for contrast. 



4. Wash and examine. 



GabbetCs Method. 1. Prepare film and stain as above with car- 

 bol-fuchsin. 



