ACID-FAST STAINING 35 



Practically all pathogenic cocci are Gram-positive, except the Gono- 

 coccus, the Meningococcus, the M. catarrhalis, and the M. melitensis. 



Practically all pathogenic bacilli are Gram-negative, except the 

 spore-bearing ones (exception B. malig. cedemat.), the acid-fast ones 

 and diphtheria and diphtheroid organisms. 



The bacillus of glanders is Gram-negative. 



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. Counterstain in saturated aqueous solution of methylene blue 

 or with Loffier'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. 



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, counters taining however, with Bis- 

 mark 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) ammo- 

 nium 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 seven 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 organisms 

 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. 



