BIOTIN 



37 



BISMUTH PIGMENTATION 



hormones and other substances adopted 

 internationally are critically considered 

 by Irwin, J. O., J. Hyg., 1950, 48, 215- 

 238. 



Biotin, see Vitamins. 



Bird's Eye Inclusions. Some of these 

 bodies, and the so-called Plimmer's 

 Bodies, seen in cancer cells are ap- 

 parently greatly enlarged Centrosomes. 

 Methods and results are given by Le- 

 Count, E. R., J. Med. Res., 1902, 7 

 (N.S. 2), 383-393. 



Birefringence, see Polarization Optical 

 Method. 



Bismark Brown Y (CI, 331) — basic brown, 

 G, GX, or GXP, Excelsior brown, 

 leather brown, Manchester brown, 

 phenylene brown, Vesuvin — A mixture 

 of basic dis-azo dj'es of different shades. 

 Quite widely employed, see Blaydes, 

 G. W., Stain Techn., 1939, 14, 105-110 

 for use with plant tissue. See Weissen- 

 berg's method, as described by McClung, 

 1950, p. 184, for the preservation in 

 sections of Bismark brown employed 

 for supravital staining of eggs. 



Bismiocymol (see Pappenheimer, A. M. 

 and Maechling, E. H., Am. J. Path., 

 1934, 10, 577-588. 



Bismuth. Microchemical detection of : 



1. Method of Christeller-Komaya. 

 Make frozen sections of formalin fixed 

 tissues. A = quinine sulphate, 1 gm.; 

 aq. dest., 50 cc; nitric acid, 10 drops. 

 B = potassium iodide, 2 gm., aq. dest., 

 50 cc. Immediately before use mix 

 equal parts A and B and add 2 drops 

 nitric acid, C.P. After treating sec- 

 tions with this for 1 min. wash very 

 quickly in 10 cc. aq. dest. + 2 drops 

 nitric acid. Mount section on slicle. 

 Dry, counterstain with gentian violet. 

 Bismuth appears as dark brown grains 

 (Lison, p. 98). See Komaya, G., Arch, 

 f. Dermat. u. Syph., 1925, 149, 277-291 

 (good colored figures) and Califano, L., 

 Zeit. f. Krebsf., 1927-28, 26, 183-190. 



2. Another modification of the 

 Komaya method is given by Castel, P., 

 Arch. Soc. d. Sci. Med. et. biol. de 

 Montpellier, 1934-35, 16, 453-456 as 

 follows : Dissolve 1 gm. quinine sulphate 

 in 50 cc. aq. dest. with aid of a few drops 

 of sulphuric acid. Dissolve 2 gm. 

 potassium iodide in 50 cc. aq. dest. 

 Mix, apply to section, gives red ppt. 

 of salts of bismuth in form of iodo- 

 bismuthate of quinine or double iodide 

 of bismuth and quinine. See Pappen- 

 heimer and Maecnling's (Am. J. Path., 

 1934, 10, 577-588) study of nuclear 

 inclusions in the kidney. 



3. Wachstein, M. and Zak, F. G., 

 Am. J. Path., 1946, 22, 603-611 have in 

 turn improved the Castel method. See 



Glick, Techniques of Histo- and Cyto- 

 chemistry, 1949, p. 31. 



Reagents: A. Modified Castel: After 

 dissolving 0.25 gm. brucine sulphate in 

 100 cc. aq. dest. add 2 gm. aq. dest. 

 Store in brown bottle and filter before 

 using. B. Diluted Castel: One part A 

 to 3 parts aq. dest. C. Levulose: Dis- 

 solve 30 gm. in 20 cc. aq. dest. at 37°C. 

 for 24 hrs. and add 1 drop of B. D. 

 Counterstain: Add 1 cc. 1% aq. light 

 green S. F. (Hartman-Leddon Co., 

 Philadelphia) to 100 cc. of B and filter 

 before use. 



Technique: Treat frozen sections or 

 deparaffinized formalin fixed sections 

 on slides for few sec. with several drops 

 30% hydrogen peroxide (Superoxol, 

 Merck) thus removing black sulfide. 

 Wash in tap water and treat with A 1 

 hr. Transfer to B and shake slightly 

 to detach precipitates. Counterstain 

 in Z) 4 min. Blot and mount in C. Bis- 

 mith appears as an orange yellow de- 

 posit. 

 Bismuth Pigmentation. Histochemical 

 identification as advised by Wachstein, 

 M. and Zak, F. G., Am. J. Path., 1946, 

 22, 603-611 depends on ability of hydro- 

 gen peroxide to decolorize bismuth 

 sulfide instantaneously and of a slightly 

 modified Castel reagent to change bis- 

 muth sulfate into an orange red deposit. 



Treat deparaffinized, or frozen, sec- 

 tions with few drops superoxol (30% 

 hydrogen peroxide, Merck) from dark 

 bottle kept in refrigerator. In a few 

 seconds black of bismuth sulfide dis- 

 appears. Wash thoroughlj' in tap wa- 

 ter and place in Coplin jar containing 

 modified Castel reagent made as fol- 

 lows: Dissolve 0.25 gm. brucine sulfate 

 (Merck or Eastman Kodak) in 100 cc. 

 aq. dest. plus 2 or 3 drops concentrated 

 sulfuric acid. Then acid 2 gm. potas- 

 sium iodide, keep in a brown bottle and 

 filrer before use. After 1 hr. transfer 

 sections to another jar containing some 

 of reagent diluted with 3 parts aq. dest., 

 and shake gently to remove precipi- 

 tates. Remove most of fluid from sec- 

 tions by blotting and cover with levu- 

 lose solution made by dissolving 30 gm. 

 levulose in 20 cc. aq. dest. at 37°C. for 

 24 hrs. to which drop of diluted Castel 

 reagent has been added. To counter- 

 stain color 4 min. with freshly filtered 

 100 cc. nondiluted reagent plus 1 cc. 

 1% aq. light green S F (Hartman-Led- 

 don Co.). 



Method also can be used for study of 

 fresh tissues and gross specimens. Add 

 cone, hydrogen peroxide drop by drop 

 to pigmented area. Decolorization is 

 rapid if bismuth sulfide is present. 

 Wash thoroughly in running water to 



