GIEMSA'S STAIN 



140 GLIA STAINING WITH ANILIN DYES 



2. For sections. Much depends upon 

 the choice of fixative. Formalin, gener- 

 ally employed in 10% solution, acts as 

 a sort of mordant for the blue component 

 so that the blue coloration is particularly 

 strong. Fixation in Regaud's gives good 

 results particularly with Rickettsia, 

 Zenker's fluid is recommended by 

 Wolbach. When this is used it is neces- 

 sary to remove the mercuric chloride by 

 treating the sections with Lugol's solu- 

 tion. They are then washed in 95% 

 alcohol and the last traces of iodine are 

 extracted by 0.5% aqueous sodium 

 hyposulphite for 10-15 min. The hypo- 

 sulphite in turn is washed out in run- 

 ning water about 5 min. and rinsing 

 in aq. dest. See Cowdry's colored 

 figures of Rickettsia, J. Exper. Med., 

 1925, 42, 231-252. Bouin's fluid (75 

 cc. saturated aq. picric acid, 26 cc. 

 commercial formalin and 4 cc. glacial 

 acetic acid) is suggested for intracellular 

 protozoa (East Coast fever parasites) 

 by Cowdry and Danks (Parasitology, 

 1933, 25, 1-63) because after Giemsa 

 staining it gives the chromatin a 

 desirable purple color (see colored 

 plate). Stain sections placed vertically 

 in staining jars in 1.5 cc. Giemsa 's 

 solution plus 50 cc. aq. dest., changed 

 during the first hour, overnight. Dif- 

 ferentiate in 95% alcohol, dehydrate 

 quickly in absolute alcohol, clear in 

 xylol and mount in balsam. 



If the sections are not blue enough add 

 1-2 drops 0.5% sodium bicarbonate and 

 1.5 cc. methyl alcohol to the stain; or 

 remove excess of mordanting potassium 

 bichromate from Zenker fixation by 

 rinsing 1 min. in 1% potassium per- 

 manganate followed by 5% oxalic acid 

 4 min. and thorough washing in aq. 

 dest., or do both. If on the contrary 

 they are too blue mordant in 5% potas- 

 sium bichromate 15 min., rinse in aq. 

 dest. until no more yellow is removed 

 and stain; or add a little colophonium 

 to the alcohol used in differentiating 

 and dehydrating of the sections, as 

 advised by Wolbach, or again do both. 



Usually Giemsa's stain gives satis- 

 factory results without any special pre- 

 cautions. The difficulty is that the 

 colors fade quite rapidly particularly 

 when the balsam is noticeably acid and 

 when the sections are left in direct 

 sunlight. Their period of usefulness 

 can be extended by mounting in cedar 

 oil, used for oil immersion objectives, 

 instead of in balsam. Try Clarite. 

 If a variety of fixatives is employed 

 it may be necessary to suit the stain to 

 the fixative by use of buffers, in which 

 case see Lillie, R. D., Stain Techn., 

 1941, 16,1-6. 



To demonstrate the "nucleoids" of 

 bacteria in smears the technique of 

 C. F. Robinow published as Addendum 

 to Dubos, R. J., The Bacterial Cell. 

 Harvard Univ. Press, 1945, 460 pp. is 

 suggested. Fix smears in osmium 

 tetroxide vapor, treat 7-10 min. with 

 N/1 HCl at 60°C. and color with 

 Giemsa's solution. Bj' this method nu- 

 cleoids are stained whereas similar 

 bacteria not treated with the acid are 

 uniformly colored by Giemsa. Robi- 

 now prefers this staining of nucleoids 

 by Giemsa after hydrolysis to the Feul- 

 gen technique. 



Gilson's Fluid. Nitric acid (sp. gr. 1.456), 

 15 cc; acetic acid, 4 cc; mercuric 

 chloride, 20 gm.; 60% ale, 100 cc; aq. 

 dest., 880 cc. Used mostly for inverte- 

 brates. 



Gilson's Mixture is equal parts chloroform 

 and cedar oil. 



Gingiva. Capillaroscopy of (McClung, Mi- 

 croscopical Technique, 1950, 328); Eo- 

 sinophile leucocytes in (Orban, B., J. 

 Dent. Res., 1940, 19, 537-543.) 



Glacial Acetic Acid, see Acetic Acid. 



Gland Cells contrasted. Endocrine, exo- 

 crine, apocrine, merocrine, holocrine, 

 serous, zymogenic and mucous (Cow- 

 dry's Histology, p. 257). 



Glass Cloth, as a substrate for tissue cul- 

 ture, Warner, D., Hanawalt, C. and 

 Bischoff, F., J. Nat. Cancer Inst., 1949, 

 10, 67-74. 



Glass Electrode. Sisco, R. C, Cunning- 

 ham, B. and Kirk, P. L., J. Biol. Chem., 

 1941, 139, 1-10 have devised an open 

 cup variety described with diagrams by 

 Click, pp. 183-184. See Claff and Swen- 

 son glass capillary electrode and the 

 Pickford sealed-in capaillary glass 

 electrode also described by Click. 



Glia Staining with Anilin Dyes (Proescher, 

 Fr., Stain Techn., 1934, 9, 33-38). 

 Fix in 10% formalin or in 90% alcohol 

 followed by formalin. Wash frozen 

 sections, 10-15 microns thick, in aq. 

 dest. Stain in sat. aq. victoria blue B 

 (not filtered but poured off from the 

 undissolved dye), 14-24 hrs. Wash 

 quickly in aq. dest., mount with glyc- 

 erin-albumen, blot and dry in air. 

 Treat with ultraviolet light 30 min. 

 Pass to N/20 iodine few sec. Remove 

 iodine, blot, dry, destain in xylol- 

 anilin, clear first in clove oil, then 

 xylol, mount in balsam. Glia blue, 

 nerve cells lightly stained, connective 

 tissue metachromatic violet or colorless. 

 Instead of ultraviolet light stained 

 sections can be treated with 0.5% 

 potassium bichromatic for 30 min. In 

 place of victoria blue, methyl violet 2B, 

 ethyl violet or crystal violet can be 

 employed. 



