64 JOHNFS DISEASE 



As has already been mentioned, Johne's bacillus 

 belongs to the acid-fast group, and is best stained by 

 the method known as Ziehl-Neelsen's. For this pro- 

 cess the following solutions are required : 



Carbol-Fuchsin. — Fuchsin, i gramme ; "Absolute 

 alcohol, 10 c.c. Dissolved and added to 90 c.c. 

 of a 5 per cent, aqueous solution of carbolic 

 acid. 



Sulphuric Acid. — A 25 percent, aqueous solution. 



Loeffler's Methylene Blue. — Saturated alcoholic 

 solution of methylene blue, 30 c.c, mixed with 

 caustic potash solution (i to 10,000 of water), 100 c.c. 



The simplest way of staining by this method is to 

 spread a thin film of the suspected material (scraping 

 of the faeces or intestine) on a slide, and dry by gently 

 heating. Then boil a little of the carbol-fuchsin solu- 

 tion in a test-tube, and pour sufficient on the slide to 

 completely cover the film (in place of carbol-fuchsin 

 carbol-gentian violet may be used). To insure that 

 the film remains covered, the slide must be left on a 

 flat bench, and should be stained for three or four 

 minutes. The stain must then be washed off with 

 water, and the film treated with sulphuric acid solu- 

 tion, the washing and acid treatment being repeated 

 until no more of the magenta colour can be extracted. 

 After washing with water, the film should be counter- 

 stained with the methylene blue solution, and this 

 washed off after a few seconds. The film should now 

 be dried, and examined under a yV oil-immersion lens ; 

 it is not necessary to use a cover-glass. 



In the case of films made from faeces, it is not easy 

 to extract all the fuchsin from certain portions of the 

 undigested food, but this in no way interferes with 

 the recognition of the bacilli, if present. By this 



