ACID ALIZARIN GREEN G 



ACID FAST BACILLI 



Acid Alizarin Green G (CI, 1049), a direct 

 mordant dye of color fastness 1. Use 

 for staining blue green and green algae 

 and paraffin sections of animal tissues 

 after mordanting in 1% aq. ferric alum 

 is described (Emig, p. 63). 



Acid Blue B (CI, 736), an acid dye of light 

 fastness 5 gives light, fugitive and in- 

 distinct coloration of tissue (Emig, 

 p. 52). 



Acid Blue G (CI, 712)— Brilliant Acid Blue 

 V — an acid dye of light fastness 5 (Emig, 

 p. 52). 



Acid Bordeaux, see Bordeaux Red. 



Acid Congo R, see Vital Red. 



Acid Dyes, see Staining. 



Acid Fast Bacilli. Of these the organisms 

 of tuberculosis and leprosy are the most 

 important. 



1. In smears apply Carbol Fuchsin 

 gently heat 3-5 min. or stain room 

 temperature 15 min.; decolorize 95% 

 ethyl alcohol containing 3% of cone, 

 hydrochloric acid until only slight pink 

 color remains; wash in water; counter- 

 stain sat. aq. methylene blue or Loef- 

 fler's Alkaline Methylene Blue; wash 

 and dry. 



2. In sections the organisms can be 

 stained red in paraffin sections after 

 almost any fixation (formalin-Zenker 

 preferred). First color with Harris 

 hematoxylin. Wash in water and per- 

 haps decolorize a little in Acid Alcohol. 

 Wash again. Stain with warmed carbol 

 fuchsin 1 hr. or more. Decolorize in 

 acid alcohol. Wash carefully in water 

 plus few drops ammonia. 95% ale, 

 abs. ale, xylol, balsam. A critique of 

 the methods has been published (Fite, 

 G. L., Am. J. Path., 1938, 14, 491-508). 

 To color the organisms blue, fix 3-5 days 

 or more in equal parts 10% formalde- 

 hyde and 95% alcohol. Stain sections 

 in new fuchsin 0.5 gm.; phenol crystals, 

 5.0 gm.; alcohol methyl or ethyl, 10 cc. 

 + aq. dest. to make 100 cc. at 60° C. 

 over night, 12-24 hrs. or at room tem- 

 perature 24-48 hrs. Longer for M. 

 leprae. Freshly distilled aq. formalde- 

 hyde 5-30%, 5 min. (Note that this 

 formalin must not be alkaline and that 

 it is safer to have it faintly acidified.) 

 2% hydrochloric acid in 95% alcohol, 

 5 min. 1% aq. potassium permanganate 

 2-5 min. (until brown). 2% aq. oxalic 

 acid, 1 min. Harris' hematoxylin 2 

 min. Stain in acid fuchsin, 0.1 gm.; 

 picric acid, 0.5 gm.; aq. dest. to make 

 100 cc. Without washing, dehydrate in 

 alcohol, clear in xylol and mount in 

 balsam. Nuclei, brown; connective 

 tissue fibers, red; muscle, yellow; acid 

 fast bacilli, dark ultramarine blue. 

 Good for photography (Fite, G. L., J. 

 Lab. & Clin. Med. 1939, 25, 743-744; re- 



vised by G. L. Fite, U. S. Marine Hos- 

 pital, Carville, La. May 13, 1946.). 



3. Mr. J. M. Albrecht employs the 

 following method in our laboratory. 

 Deparaffinize 5-6 n sections of 10% 

 formalin or Regaud fixed tissues. Wipe 

 off excess water around sections and 

 cover with strip of filter paper. Flood 

 filter paper with carbol fuchsin (Phenol 

 crystals, 8 gm.; basic fuchsin, 4 gm.; 

 95% ethyl alcohol, 20 cc; aq. dest., 100 

 cc). Steam for 3 min. and then allow 

 to stand for 30 min. adding more stain 

 if necessary. The filter paper prevents 

 deposition of ppt. of dye on sections. 

 Flush off stain with aq. dest. Partly 

 differentiate in 1 cc. cone hydrochloric 

 acid in 100 cc. 70% alcohol, sections be- 

 coming deep pink. Wash in aq. dest. 

 Stain Harris' Hematoxylin 10 min., 

 wash in aq. dest. Complete differentia- 

 tion of both fuchsin and hematoxylin in 

 50 cc. 70% ale -f 4-5 drops hydrochloric 

 acid, sections becoming light pink. 

 Wash in aq. dest. Neutralize in 6 drops 

 cone ammonia + 50 cc. aq. dest. 

 Wash, dehydrate, clear and mount as 

 usual. 



4. In frozen sections (Krajian, A. A., 

 Am. J. Clin. Path., Techn. Suppl., 1943, 

 7, 45-47). Transfer frozen sections of 

 leprous tissue to slides. Dehydrate, 

 blot with filter paper, dip in celloidin. 

 Blow over surface till dry. Wash in tap 

 water. Apply Carbol Fuchsin steaming 

 gently for 3 min. Pour off and wash in 

 tap water. Differentiate with 1 gm. 

 arsenic acid in 100 cc. 60% alcohol ap- 

 plied by medicine dropper. Again wash 

 in tap water and counterstain with 

 Loeffler's methylene blue 2 min. Wash 

 in tap water, dehydrate with 3 applica- 

 tions of anhydrous isopropanol or 

 absolute ethyl alcohol. Apply imme- 

 diately equal parts anhydrous iso- 

 propanol or abs. alcohol and beechwood 

 creosote. Agitate slide removing ex- 

 cess blue color. Blot with filter paper, 

 clear with xylol and mount in damar. 



See Tubercle and Leprosy Bacilli, 

 Fluorescence Microscopy, also paper by 

 Richards, O. W., Kline, C. K. and 

 Leach, R. E., Am. Rev. Tubere, 1941, 

 44, 255-266. Efficiency of Ziehl-Neel- 

 sen and fluorescence techniques com- 

 pared. The latter superior (Van Dyke, 

 A. E., Am. J. Clin. Path., Techn. 

 Suppl., 1943, 7, 6-8.) For acid fast 

 bacilli in urine see Kelso, R. E. and 

 Galbraith, T. W., Am. J. Clin. Path., 

 Techn. Suppl., 1943, 7, 8-11. 



Less is known about the conditions 

 that determine acid fastness than those 

 which determine Gram positiveness 

 (see Gram Stain). The facts are well 

 stated for mycobacteria in general and 



