ACID FAST BACILLI 



18 



ACID RUBIN 



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. A brecht 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 + 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. Tuberc, 1941, 

 44, 255-266. Efiiciency 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 

 especially for the Tubercle Bacillus by 

 Dubos, R. J., The Bacterial Cell. 

 Harvard Univ. Press, 1945, 460 pp. 

 There is present in the tubercle bacillus 

 mycolic acid which is acid fast even 

 after isolation in the pure state; but 

 the property of acid fastness is lost by 

 the bacilli under conditions that do not 

 destroy this acid. These conditions 

 involve destruction or impairment of 

 structure of the organisms by mechani- 

 cal, chemical or enzymatic means. 

 Apparently the cell surface must be 

 intact. Dubos quotes Yegian et al. as 

 showing that tubercle bacilli stained in 

 absence of electrolytes are uniformly 

 colored rods, that addition of electro- 

 lytes causes a beaded appearance and 

 that treatment with ethyl alcohol re- 

 stores uniform solid staining to beaded 

 organisms which means that the change 

 from beaded to uniform state is a re- 

 versible process. This dependence of 

 microscopic appearance on experi- 

 mental conditions of technique is ob- 

 viously a matter of great consequence 

 in leprosy as well as in tuberculosis. 

 The investigator has to check carefully 

 by study of living unstained bacilli. 



Acid Fuchsin (CI, 692) — acid magenta, acid 

 rubin, fuchsin S, SN, SS, ST or S Ill- 

 Commission Certified. Since this is a 

 sulfonated derivative of basic fuchsin, 

 and, because there are 4 possible pri- 

 mary basic fuchsins, Conn (p. 118) points 

 out that at least a dozen primary acid 

 fuchsins are possible and samples are 

 usually mixtures of several. Acid 

 fuchsin is employed is so many ways 

 that to enumerate them would be both 

 futile and unnecessary. See New 

 Fuchsin. 



Acid Green, see Light Green SF yellowish. 



Acid Green O, see Naphthol Green B. 



Acid Hemalum, see Hemalum. 



Acid Magenta, see Acid Fuchsin. 



Acid Orange II, Y or A, see Orange II. 



Acid Phloxine GR, see Chromotrope 2R. 



Acid Rubin, see Acid Fuchsin. 



