STAINS, ETC. 



1388 



STAINING OF MICROORGANISMS 



downward) upon a watch-glassful of Ehrlich-Weigert's 

 anilin-methyl-violet stain ; heat the watch-glass gently 

 until the fluid begins to steam ; let it cool for 3 to 5 

 minutes ; decolorize in I part nitric acid and 3 parts 

 water, and then in 60 per cent, alcohol for I or 2 

 seconds; wash in water; counterstain with a saturated 

 aqueous solution of Bismarck-brown ; wash, dry, and 

 mount. Anilin-gentian-violet or anilin-fuchsin may be 

 used instead of anilin-methyl-violet. Sections of tissue 

 should not be heated. Gram's Method. Heat 2 to 

 5 minutes, or stain cold 20 to 30 minutes (tubercle- 

 bacilli, 12 to 24 hours), in saturated solution of gentian- 

 violet anilin-water ; rinse quickly in absolute alcohol; 

 transfer to Gram's solution (1 to i}4. minutes), in which 

 the specimen turns black ; wash in alcohol until the 

 black color vanishes and a pale-gray color appears ; 

 dry and mount in Canada balsam. The decolorization 

 may be hastened by adding 3 per cent, nitric acid 

 to the alcohol and then washing in pure alcohol. All 

 the tissue-cells are decolorized by this method, while 

 the bacteria are stained a deep-blue. The cells may be 

 subsequently stained with a watery or alcoholic solution 

 of Bismarck-brown 2 to 5 minutes, then washed in 

 absolute alcohol until the section is yellowish-brown. 

 This method is of diagnostic value, as certain bacteria 

 are stained, others decolorized by it. Bacteria stained 

 by Gram's method : tubercle-bacillus ; Fraenkel- Weich- 

 selbaum pneumococcus ; streptococcus pyogenes ; 

 streptococcus of erysipelas ; staphylococcus pyogenes 

 aureus, albus, citreus, and flavus ; anthrax-bacillus ; 

 bacillus of hog-erysipelas. Bacteria decolorized by 

 Gram's method : Typhoid- bacillus, gonococcus, Fried- 

 lander's capsule-bacillus, Koch's comma-bacillus, 

 glanders-bacillus, and the spirillum of relapsing fever. 

 Botkin advises washing the preparation in plain anilin- 

 water before decolorizing in the iodin-solution. Modifi- 

 cations of Grant's Method. I. Weigerfs. The sec- 

 tions, stained with gentian- violet, or methyl-violet, are 

 not transferred to alcohol from the iodin-solution, but 

 are laid upon slides and covered with anilin-oil. 

 This is removed with blotting-paper, and followed 

 by xylol and xylol-balsam. The anilin-oil dehydrates 

 and differentiates. 2. /Cuhne's. (a) A solution is pre- 

 pared of 1 gram of Victoria blue in 50 c.c. of 50 

 per cent, alcohol, and diluted to half its strength with 

 0.5 per cent, aqueous solution of ammonium car- 

 bonate. Stain from I to 5 minutes ; decolorize in 

 Gram's solution, and treat as in Gram's method, using 

 fluorescein (l gm. to 50 c.c. absolute alcohol) instead 

 of alcohol, to extract the stain. (t>) Add hydrochloric 

 acid (I drop to 50 gm. w T ateh to a concentrated aque- 

 ous solution of violet, and use for staining ; continue as 

 in Gram's method. 3. Giinther's (Schetik). The 

 cover-glass preparation or the section is left for about 



2 minutes in gentian-violet anilin-water. Excess of 

 stain is removed with blotting-paper, and the specimen 

 brought for 2 minutes into Gram's solution, then into 

 pufe alcohol for half a minute, exactly 10 seconds into 



3 per cent, hydrochloric acid in alcohol, then directly 

 into fresh, plain alcohol, which is changed until no 

 more color is extracted from the preparation. Cover- 

 glass preparations are dried and mounted ; sections 

 cleared in xylol and mounted in xylol-balsam. Tubercle- 

 bacilli and lepra-bacilli must remain in the staining fluid 

 12 hours. Koch's Method, for bacteria in tissues. 

 Stain in aqueous solution of methyl-violet, fuchsin, or 

 methylene-blue. Wash in a saturated solution of ]x>tas- 

 sium carbonate diluted with an equal volume of water. 

 The color will be removed from the nuclei x>f the cells, 

 but remains in the bacteria; dehydrate, clear in cedar- 

 oil, and mount in balsam. Kuhne's Carbol-methyl- 

 ene-blue Method. The section is placed in the fol- 



lowing solution for about )/ 2 hour: methylene-blue 1.5 

 parts, absolute alcohol 10 parts; triturate in a watch- 

 glass and add, gradually, 100 parts of a 5 per cent, solu- 

 tion of carbolic acid. After staining, wash the section 

 in water; decolorize carefully in hydrochloric acid, 10 

 drops to 500 c.c. of water ; immerse at once in a solution 

 of lithium (8 drops of saturated solution of lithium car- 

 bonate in 10 c.c. of water) ; place in a bath of distilled 

 water for a few minutes ; dip into absolute alcohol 

 colored by methylene-blue ; dehydrate in anilin-oil that 

 contains a little methylene-blue in solution : wash in 

 pure anilin-oil (not colored), then in alight fluid ethereal 

 oil, as oil of thyme or terebene; clear in xylol; mount 

 in balsam. Recommended for staining the bacillus of 

 glanders. Kuhne's Dry Method. Sections are stained 

 for 10 to 15 minutes in I per cent, solution of ammonium 

 carbonate mixed with a concentrated aqueous solution 

 of methylene-blue; then wash in water, decolorize in 

 an aqueous solution of hydrochloric acid ; wash in 

 water, dry upon the slides, clear in xylol, and mount in 

 balsam. Pregl's Substitute for Kuhne's Method. 

 The sections, fixed to a slide or cover-glass by Pregl's 

 method (see Fixatives) , are stained by dropping Kuhne's 

 carbol-methylene-blue solution upon them. Wash in 

 water, and place immediately in 50 per cent, alcohol. 

 When the sections acquire a pale-blue color with a green- 

 ish tinge, dehydrate in absolute alcohol, clear in xylol, and 

 mount. Unna's Methods. For the recognition of 

 fungi in epidermic scales. I. Place the crust or comedo 

 on a slide, add a drop of acetic acid, and rub to a pulp 

 by means of another slide laid crosswise upon the first; 

 separate the slides, and dry quickly over a spirit-flame. 

 Wash out the fat with a few drops of ether and alcohol, 

 and stain in two drops of solutions of borax and methyl- 

 ene-blue, cover with the other slide, and warm over the 

 flame for 10 to 20 seconds ; rinse in water, decolorize in 

 glycol 2 to 5 minutes, rinse again in water, then in alcohol, 

 dry over the flame, and mount in balsam. 2. Treat sections 

 for 5 minutes with an aqueous solution of borax -methyl- 

 ene blue, then for 5 minutes with a 5 per cent, solution 

 of potassium iodid to which a crystal of iodin has been 

 added. Rinse in alcohol until a blue cloud forms ; 

 differentiate in creasote for from a few seconds to half an 

 hour, according to the intensity of the stain. Carry to 

 rectified oil of turpentine. In this the bluish color changes 

 to red or brown. Preserve in a solution of colophon- 

 ium in oil of turpentine. Weigert's Iodin Method 

 (Schenk). Sections are stained in gentian-violet anilin- 

 water, rinsed in a solution of common salt, laid upon 

 the slides, and dried ; then solution of iodin is dropped 

 upon them. After again drying, anilin-oil is poured 

 over the sections, renewed several times, and finally 

 displaced by xylol. Mount in balsam. II. SPECIAL 

 Methods. Actinomyces. Select whitish granules 

 from the suspected pus by spreading it on a glass 

 upon a dark background. Prepare cover-glasses a> for 

 other methods. The preparation may be stained in 

 lithium-carmin and then by Weigert's method fot 

 bacteria. I. YVeigert advises a dark-red solution ol 

 orseille in absolute alcohol 20 parts, acetic acid 5 parte, 

 and water 40 parts. The specimen is left in the stain 

 I hour, then washed in alcohol and counterstaitttd 

 in I per cent, aqueous solution of gentian-violet, h 

 is again washed in alcohol, and mounted. 2. Gram 1 

 Method, with eosin as a contrast-stain, shows the 

 clubs very well. 3. Ehrlich 's -Method [Crookshank). 

 Sections are placed in alcohol or distilled water, then 

 in Ehrlich's logwood for about % minute, and • 

 ferred to distilled water; then placed in a large dish ol 

 tap- water, y 2 hour or more, till a blue color ap- 

 pears. They are next stained in a solution of nil in S. 

 and orange, I to 2 minutes; washed in distilled water, 



