GOSSYPIMINE 



149 



GRAM'S STAINS 



aq. silver nitrate until ppt. is dissolved. 

 Add 5 cc. 3.1% aq. soaium hydroxide 

 and redissolve ppt. with strong 

 ammonia. With aq. dest. dilute to 100 

 cc). Wash in aq. dest. at 60 °C. Re- 

 duce in: 10% formalin 90 cc. + 2.5% 

 iron alum 10 cc. Wash in tap water, 

 dehydrate in alcohol, clear in xylol 

 and mount in balsam. 



Gossypimine, see Safranin O, 



Grafts. Intracoelomic of eye primordium, 

 Joy, E. A., Anat. Rec, 1939, 74, 461- 

 486. See Transplantation. 



Gram, see Weight measurements. 



Gram's Iodine Solution. Iodine, 1 gm.; 

 potassium iodide, 2 gm.; aq. dest., 

 300 cc. A stronger solution may be 

 desirable with only 100 cc. aq. dest. 



Gram-Pappenheim stain as modified for 

 smears and paraffin sections (Scudder, 

 S. A., Stain Techn., 1944, 19, 39-44). 



Gram's Stains for bacteria: 



1. In smears. Hucker modification 

 (McClung, p. 138). Stain 1 min. in 

 equal parts A and B : A = crystal violet 

 (85% dye content, 4 gm.; 95% ale, 20 

 cc.) B = ammonium oxalate, 0.8 gm.; 

 and aq. dest. 80 cc. After washing in 

 water immerse in: iodine, 1 gm. potas- 

 sium iodide, 2 gm., aq. dest., 300 cc. 

 1 min. Then wash in water and dry 

 by blotting. Decolorize 30 sec. in 95% 

 ale. gently moving. Blot and counter- 

 stain in: 10 cc. sat. safranin in 95% 

 ale. and aq. dest. 100 cc. Wash and dry. 

 Kopeloff-Beerman Modification (Mc- 

 Clung, p. 139). Stain 5 min. or more 

 in: 1% aq. gentian or crystal violet, 1.5 

 cc. mixed before use with 0.4 cc. 5% 

 aq. sodium bicarbonate. Rinse in iodine 

 solution made by dissolving 2 gm. 

 iodine in 10 cc. normal sol. sodium 

 hydroxide and adding 90 cc. aq. dest. 

 and stand 2 min. or more. Blot dry. 

 Add 100% acetone drop by drop with 

 specimen tilted till no more color is 

 removed, less than 10 sec. Dry in air. 

 0.1% aq. basic fuchsin, 10-30 sec. 

 Wash in water and dry. Weiss Modifica- 

 tion (Weiss, E., J. I^ab. & Clin. Med., 

 1940-41, 26, 1518-1519). Make thin, 

 uniform smears and fix over flame. 

 Cover slide with 3% gentian violet in 

 20% alc^ 3-5 min. Wash in warm 

 water. Cover 3-5 min. with iodine, 20 

 gm. ; potassium iodide, 40 gm., aq. dest. 

 300 cc. Wash with warm water. De- 

 colorize in acetone and wash imme- 

 diately in water. Counterstain quickly 

 in 2% basic fuchsin in 95% ale. Wash 

 in water, drj-^ and examine. 



The use of colloidal iodine has been 

 suggested to improve the reaction be- 

 tween bacteria and stain (Lyons, D. C, 

 J. Lab. & Clin. Med., 1936^37, 22, 

 523-524). Methods for preparing col- 



loidal iodine are described by Chandler 

 and Miller (W. L. and E. J., J. Phys. 

 Chem., 1927, 31, 1091-1096). 



2. In sections. Grarn-Weigert method 

 (McClung, p. 152). Fix in Zenker's 

 fluid. Stain paraffin sections lightly in 

 alum hemato.xylin and wash in running 

 water. 1% aq. eosin, 1-5 min., followed 

 by washing in water. Stain ^-1 hr. in 

 anilin methyl violet made by mixing 1 

 part of A with 9 of B : A. abs. ale. 33 cc. ; 

 aniline oil, 9 cc. ; methyl violet in excess. 

 B. Saturated aq. methyl violet and wash 

 in water. Lugol's iodine 1-2 min. and 

 wash in water. Blot; dehydrate and 

 clear in equal parts aniline oil and 

 xylol several changes. Wash with xylol 

 and mount in balsam. Glynn's method. 

 (Glynn, J. H., Arch. Path., 1935, 20, 

 896-899). To make stain triturate 1 

 gm. crystal violet and 1 gm. phenol 

 crystals in mortar and add 10 cc. 

 absolute alcohol. Before using dilute 

 10 times with aq. dest., allow to stand 



2 days and filter. Stain deparaffinized 

 sections of Zenker (less acetic), Bouin, 

 Helly or 10% formalin fixed material 

 for 2 min. Drain off but do not wash. 

 Add Gram's iodine, 1 min. Differentiate 

 in acetone until no more color is given 

 off, 10-15 sec. Wash in aq. dest. 

 Counterstain in 0.05% basic fuchsin 

 in N/500 hydrochloric acid (see Normal 

 Solutions). Drain, do not wash, apply 

 1% aq. trinitrophenol, j-l min. Wash 

 in aq. dest. Dehydrate and differen- 

 tiate in acetone 10-15 sec, clear in xylol 

 and mount in balsam. Gram -f bac- 

 teria, violet; Gram—, red; nuclei, light 

 red; cytoplasm, yellow. 



3. For organisms in frozen sections 

 by Krajian, A. A., Arch. Path., 1941, 

 32, 825-827. Stain 7-10m frozen sec- 

 tions for 2 min. in Harris' alum hema- 

 toxylin. Wash in tap water till blue 

 and destain quickly by dipping 5 to 7 

 times in acid alcohol. Rinse in tap 

 water and apply following solution for 



3 min. — copper sulfate, 7 gm.; zinc sul- 

 fate, 4 gm. dissolved in 100 cc. aq. with 

 aid of heat. Pour off and apply 0.3 gm. 

 brilliant green in 10 cc. above copper 

 zinc mixture for 5 min. Rinse in water 

 and fortify with 5% aq. ammonium ni- 

 trate for 1 min. Rinse in tap water and 

 stain with carbol fuchsin (Ziehl-Neel- 

 sen) for 2 min. Rinse in tap water, blot 

 and apply dioxane for 2 min. Pour off 

 and add equal parts creosote and .xylol, 

 changing tliis mixture and agitating to 

 promote even differentiation until back- 

 ground appears clear red. Clear in 

 pure xylol (2 min.) and mount in damar. 

 Gram positive organisms bluish green; 

 gram negative ones red. 



The Gram staining technique and 



