STAINING I Q 



BISMARCK BROWN 



Bismarck brown, 0.2 Gm. 



Boiling distilled water, 100 cc. 



Mix together and filter. 



Most bacteria when stained can be decolorized by washing with acids. 

 But there are some bacteria that cannot be decolorized by acids after they have 

 been stained. These we call acid-fast. A number of organisms which are 

 acid-fast can be decolorized with alcohol. They are acid-fast, but not alcohol- 

 fast. Of the pathogenic organisms there are but two which are both acid-fast 

 and alcohol-fast, the tubercle bacillus and the leprosy bacillus. 



It has been recently stated that there are non-acid fast and non-alcohol- 

 fast strains of the leprosy bacillus. Practically, the tubercle bacillus is always 

 acid- and alcohol-fast; but the degree of resistance to acid and alcohol of some 

 other organisms at times simulates that of the tubercle bacillus, so that rare 

 occasions arise when differentiation can only be determined by cultural test. 



To demonstrate acid-fast bacilli, apply carbol fuchsin for 15 minutes, wash 

 with 25 per cent, aqueous solution sulphuric acid until specimen gives off no 

 more color, wash with water, apply LoefHer's methylene blue for 3 to 5 minutes, 

 wash in water, dry and examine. 



When the carbol fuchsin is applied everything is stained red. The acid 

 removes the stain from all but the acid-fast organisms, these remain red, the 

 others are decolorized. When the methylene blue is added it does not affect 

 the acid-fast organisms, they are already saturated with fuchsin and remain 

 red, but it tints everything else blue, so that the acid-fast organisms appear red 

 and the non-acid-fast organisms blue. 



Bacteria that are decolorized by acids are also decolorized by alcohol. 



To distinguish bacteria that are acid-fast but not alcohol-fast, from those 

 which are both acid- and alcohol-fast, make several slides, stain some with 

 carbol fuchsin 5 to 15 minutes, apply 25 per cent, aqueous solution of sulphuric 

 acid for 2 or 3 minutes, wash with water and apply LoefBer's methylene blue. 

 Acid-fast organisms will appear red. The remaining slides stain with carbol 

 fuchsin as before, then apply either absolute alcohol or 5 per cent, alcoholic 

 solution of sulphuric acid or 3 per cent, alcoholic solution of nitric acid for 10 

 to 15 minutes. Wash with water and counter stain with LoefHer's methylene 

 blue. Bacteria that are both acid-fast and alcohol-fast will appear red, those 

 which are acid-fast but not alcohol-fast will appear blue. 



In 'diagnostic work, when examining sputum, etc., for tubercle bacilli, time 

 and labor are commonly saved by combining counter stain and decolorizing 

 agent, as in Gabbet's solution and Pappenheim's solution, which are the ones 

 commonly used. After staining with carbol fuchsin 5 to 15 minutes, Pappen- 

 heim's solution is applied for 15 minutes, then wash in water, dry and mount. 

 Organisms which are both acid- and alcohol-fast, appear red, the others blue. 



When Gabbet's solution is used it is applied for 2 minutes, after staining 

 with carbol fuchsin. The specimen is then washed with water, dried and ex- 



