476 METHODS AND FORMULAS DS 23.213 



S3.S1S Methods for Acid-fast Organisms 



"Acid-fast" bacteria were originally so identified because a solution of magenta was not re- 

 moved from them by strong acid. These techniques are commonly all lumped together as 

 " Ziehl-Neelsen techniques" for the reason that the phenol-magenta stain of Ziehl 1882 was in 

 1883 used for the first demonstration of acid-fast bacteria by Neelsen. It is nowadays, how- 

 ever, most unusual to use so violent an acid solution (25% nitric acid) as did Neelsen, and 

 there is no justification for the blanket retention of the names of these two technicians for 

 every acid differentiated stain which has been proposed. Many, if not indeed most, of the 

 modern techniques do not use acid for differentiation at any stage of the proceedings, and 

 attention should be drawn to the technique of Burke, Dickson, and Phillips 1932 by which 

 acid-fast bacteria may be clearly differentiated from all other bacteria in the section without 

 the use of any acid at all. Similarly, the technique of Fontes 1909 gives a similar differentia- 

 tion using neutral solution. When the term "Ziehl-Neelsen" is loosely used in the literature 

 today, the technique of Giinther 1899 is the one which is most usually intended. 



23.213 Alexander 1932 19938, 75:197 



REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 3% nitric acid in 70% ale; C. DS 11.44 



Loffler 1890; D. 0.05% sodium hydroxide 

 method: [heat- fixed smears] -^ A, on slide, warmed to steaming, 5 mins. — > rinse — > C, 



on slide — * D, 6-8 drops, added to C on slide, 2-3 mins. -^ wash -^ dry 



23.213 Alexander and Jackson 1944 19938, 99 :307 



REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. 3% hydrochloric acid; C. DS 11.44 



Loffler 1890; D. 4% sodium hydroxide; E. 0.2% sodium hydrosulfite; F. water 100, 



light green 0.5, fast yellow 0.5 

 method: [heat-fixed smear] —> ^, 3 mins. -^ wash —> B, 1-3 mins. —» wash —> C, on 



slide -^ D, 6 drops added to C on slide, 1 min. -* E, flooded on slide — > thorough wash 



— > F, flooded on slide — * wash —> dry 



23.213 Augusta 1932 6630, 11:719 



REAGENTS required: A. DS 11.43 Ziehl 1882 50, DS 12.15 crystal violet (Auguste) 50; 



B. 95% ale. 60, acetic acid 30, picric acid to sat.; C. 1% methylene blue 

 method: [heat-fixed smears] -^ A, warmed to steaming, 3 mins. — * B, till no more color 

 comes away — » water, wash -^ C, 15 sees. -^ water, wash —* dry 



23.213 Blot 1901 see DS 23.213 Neelsen 1883 (note) 



23.213 Burke, Dickson, and Phillips 1932 20540b, 7:21 



REAGENTS REQUIRED: A. DS 11.43 Ziehl 1882; B. sat. sol. malachite green in acetone 

 method: [smears] — > A, on slide, warmed to steaming, 3-5 mins. -^ water, rinse — + B, 



flood on slide and replaced as evaporation occurs, 3-5 mins. —^ wash — » dry 

 result: acid-fast bacteria, red; others, green. 



23.213 DogUo 1932 20540b (abstr. 1933) 8:76 



reagents required: ^4. DS 11.43 Ziehl 1882; B. water 85, 95% ale. 20, sulfuric acid 10, 



brilliant yellow 0.15 

 method: [heat-fixed smears] -^ A, warmed to steaming, 2-3 mins. -^ thorough wash — » 

 B, 40-50 sees. — > wash — * dry 



23.213 Fontes 1909 13465,1:59 



reagents required: A. DS 11.43 Ziehl 1882; B. DS 12.15 crystal violet; C. ADS 12.2 



Lugol (1905); D. abs. ale. 60, acetone 30; E. 1% methylene blue 

 method: [heat-fixed smears] — * A, warmed to steaming 2 mins. — > wash — > B, warmed 



to steaming, 2 mins. —* C, mixed with B on slide, 1 min. -^ D, till no more color comes 



away -^ water, wash -^ E, 15 sees. -^ water, wash -^ dry 



23.213 Fraenkel 1884 2813, 13:1 



reagents required: A. DS 23.211 Ehrlich 1882; B. water 50, ale. 30, nitric acid 20 



methylene blue to sat. 

 method: [heat-fixed smears] —» A, 12 hrs. -^ water, rinse —> B, dipped until smear 



changes from blue to red -^ water, wash —>■ dry 



