ACID-FAST STAINING 41 



Method for Staining Acid-fast Bacilli. i. Carbol fuchsin, with gen- 

 tle steaming for three to five minutes or in the cold for fifteen minutes. 



2. Wash in water. 



3. Decolorize in 95% alcohol containing 3% of hydrochloric acid 

 (acid alcohol), until only a suggestion of pink remains almost white. 



4. Wash in water. 



5. Counters tain in saturated aqueous solution of methylene blue or 

 with Loffler's methylene blue. 



6. Wash, dry, and mount. 



The steaming of the slides with carbol fuchsin is most conveniently carried out 

 by resting the slides on a piece of glass tubing bent into a V or U shape. 



The Leprosy Bacillus is usually considered as being rather easily decolorized by 

 alcohol. It is therefore often recommended to use 20% aqueous solution of sul- 

 phuric acid or nitric acid for decolorization instead of the acid alcohol above recom- 

 mended for tubercle bacilli. I have often found the leprosy bacilli as resistant to 

 alcohol as tubercle bacilli. The smegma bacillus, however, easily decolorizes with 

 the acid alcohol and in a well-decolorized smear from urinary sediment one can 

 usually feel sure that any acid-fast bacilli are tubercle bacilli. 



Fontes Method. A method in which the organisms or granules which stain by 

 the Gram method, and to which so much importance is attributed by Much, may 

 be stained, as well as those retaining acid-fast properties, has been proposed by 

 Fontes. The method is to stain the preparation with carbol fuchsin, decolorize 

 with acid alcohol, then carry through the various steps of the Gram method, 

 counterstaining, however, with Bismark brown. Fontes in his method used i part 

 of absolute alcohol and 2 parts of acetic acid as the decolorizing agent. I have 

 obtained, however, just as satisfactory results with the acid alcohol. By this 

 method the acid-fast tubercle bacilli show as red rods dotted with violet granules. 

 Those which do not fully retain acid-fast properties show as zigzag violet lines. 



Herman's Stain for Tubercle Bacilli. It has been claimed that this stain gives 

 better satisfaction than the Ziehl-Neelsen. It consists of two solutions: (i) am- 

 monium carbonate in distilled water, i%; (2) crystal violet (methyl violet 6B) in 

 95% ethyl alcohol, 3%. The two solutions are kept in separate bottles and, for 

 staining, i part of (2) is mixed with 3 parts of (i). The sections are placed on a 

 cover-glass, the water evaporated, and about 7 drops of the staining mixture 

 are placed on the specimen and allowed to steam for one minute over a water-bath. 

 Place for a few seconds in 10% nitric acid and then in 95% alcohol to decolorize. 

 Mount without a counterstain or use eosin i% or a very dilute fuchsin. The organ- 

 isms are purple. This staining method may be applied to smears of concentrated 

 or unconcentrated sputum in the same manner as for sections of tissue. 



Smith's formol fuchsin : 



Saturated alcoholic solution basic fuchsin 10 c.c. 



Methyl alcohol 10 c.c. 



Formalin 10 c.c. 



Distilled water to make 100 c.c. 



