STAINS, ETC. 



1390 



STAINING OF MICROORGANISMS 



phuric acid ioo c. c. and methylene-blue 2 c. c. , for 

 I or 2 minutes ; wash in water ; dry and mount. In 

 the case of sections, dehydrate with alcohol ; clear and 

 mount. A good and quick method. 7. abbes' Method 

 (Schenk). Two grams of fuchsin and one of methylene- 

 blue are slowly introduced into a solution of 3 c. c. of 

 anilin-oil in 15 c.c. of absolute alcohol. When they 

 are completely dissolved, 15 c.c. of water are added. 

 A few drops are heated in a test-tube and poured into 

 a watch-glass. In this the cover-glass is laid for 5 

 minutes, then washed in alcohol until no more color is 

 given off. The bacilli appear red on a blue ground. If 

 desired, stain further with a concentrated aqueous solu- 

 tion of eosin. 8. Giinther' s Method (Schenk). Stain 

 with warm anilin- water- fuchsin ; carry the cover-glass, 

 with the prepared side up, into alcohol acidulated with 

 hydrochloric acid, 3 : 100 ; move about for a moment 

 and rinse in water. With a pipet, let a few drops' of a 

 dilute alcoholic solution of methylene-blue fall upon the 

 cover-glass ; wash in water, dry, pass three times over 

 the flame, and mount in xylol-balsam. 9. Kaatzer' 's 

 Method. Float the cover-glass preparation for 24 

 hours in a supersaturated alcoholic solution of gentian- 

 violet, or, if warmed to 8o° C, for 3 minutes. De- 

 colorize in a solution of 90 per cent, alcohol 100 

 c.c, water 20 c.c, strong hydrochloric acid 20 

 drops. Rinse in 90 per cent, alcohol, after-stain for 2 

 minutes in concentrated aqueous solution of vesuvin, 

 wash in distilled water, dry, and mount in balsam. 

 10. Koch's Original Method [Sternberg). Stain sec- 

 tions or cover-glass preparations in Koch's methylene- 

 blue solution, containing caustic potash, for 20 to 24 

 hours, or, if heated to 40 C, for ^ to 1 hour ; then 

 in an aqueous solution of vesuvin for 2 minutes. 

 Wash out excess of stain in water. Dry the cover- 

 glass preparation in air and mount. Dehydrate the 

 sections in absolute alcohol, clear in cedar-oil, mount 

 in balsam. The bacilli are stained blue and the 

 tissues brown. This method has been superseded by 

 the Koch-Ehrlich Method. II. Koch- Ehrlich Method 

 [Schenk). Stain" the cover-glass preparation for 24 

 hours at the temperature of the room, or for 15 minutes 

 with heat, in anilin-water alcoholic solution of fuchsin, 

 gentian-violet, or methyl-violet (prepared by adding to 

 anilin-water enough of a concentrated alcoholic solu- 

 tion of the dye to produce opalescence). Then 

 immerse the preparation in ^^ per cent, nitric acid, 

 and when it assumes a yellow-green tint transfer to 70 

 per cent, alcohol. Counterstain I to 5 minutes in 

 methylene-blue, malachite-green, or picric acid for fuch- 

 sin, in Bismarck brown for gentian-violet or methyl- 

 violet. Wash in water, dry, and mount in balsam. 

 Prolonged action of the nitric acid will decolorize the 

 bacilli. 12. Pfuhl- Petri's Method (Schenk). Prepara- 

 tions are stained in 10 c.c. of an alcoholic solution of 

 fuchsin in 100 c.c. of water, decolorized in glacial 

 acetic acid, washed in water, dried, and mounted in 

 balsam. 13. Pittion's Method (Schenk). The pre- 

 pared cover-glass is dipped for a minute into a mixture 

 of an alcoholic fuchsin-solution I part, and 3 per cent, 

 ammonia IO parts, rinsed in water, carried to a con- 

 centrated solution of anilin green in alcohol 50 c.c, 

 water 30 c.c, nitric acid 20 c.c. for ^ of a minute, 

 rinsed, dried, and mounted. 14. Peeves' (J. E.) 

 Method. A carbolized, double stain, used for either 

 tissue or cover-glass preparations, is made as fol- 

 lows: Dissolve 2.5 gm. of rosanilin hydrochlorate and 

 Ii5 gm. of methylene-blue in 20 c.c. of absolute alco- 

 hol and 3 c.c. of anilin-oil ; agitate thoroughly, and 

 gradually add 30 c.c. of 5 per cent, carbolized water. 

 Filter a little of the stain into a watch-glass, heat it 

 gently until it steams ; drop enough of the hot stain on 



the cover-glass preparation to flood it, and cover it with a 

 bell-glass for 5 to 10 minutes. Wash and decolorize in 

 alcohol, dry, and mount in xylol-balsam. Fifteen to 

 twenty minutes are required to stain sections ; otherwise 

 the procedure is the same as for cover-glass preparations. 

 15. Tubercle-bacilli in Milk (Schenk). Place a drop of 

 the milk upon a cover-glass, add 2 or 3 drops of a 1 per 

 cent, solution of sodium carbonate, and mix well with a 

 platinum needle ; then warm carefully until complete 

 evaporation has taken place. A thin film of soap 

 remains, and is stained like an ordinary cover-glass 

 preparation. Unna's Method. Stain for from 10 

 minutes to several hours in polychromic methylene-blue 

 (Griibler), wash in water, and treat with a 33 per cent, 

 aqueous solution of tannic acid 2 to 5 minutes. Wash 

 thoroughly and transfer to absolute alcohol, gold- 

 orange alcohol, or to 25 per cent, nitric acid ; follow 

 with dilute alcohol, water, and absolute alcohol. Clear 

 in bergamot-oil. This process double-stains tubercle- 

 bacilli and leprosy-bacilli. 16. Weichselbauni 's modifica- 

 tion of the Ziehl- Neelsen Method (Schenk). The red- 

 stained cover-glass preparations are transferred directly 

 to an alcoholic methylene-blue solution, in which they 

 remain until they show a homogeneous blue color. They 

 are then rinsed in water, dried, and mounted in balsam. 

 17. Ziehl- Neelsen Method. Float the cover-glass pre- 

 paration upon Ziehl's carbol-fuchsin, heat till vapor 

 arises (about 3 to 5 minutes), wash in water, ami 

 colorize in 1 5 per cent, nitric or 5 per cent, sulphuric 

 acid, then in 60 to 80 per cent, alcohol to remove the 

 remnant of color. Wash well, dry, and mount in I 

 balsam. In the case of tissue-sections, stain cold for 

 15 minutes and decolorize as detailed ; upon removal | 

 from the alcohol, counterstain with methylene-blue;! 

 wash, dehydrate, clear, and mount. Bacillus typhi j 

 abdominalis. Stains with anilin dyes, but is easily 

 decolorized. Does not stain by Gram's method. Cover- j 

 glass preparations stain well with aqueous solutions, 

 especially fuchsin. They are to be rinsed in water,! 

 not in alcohol. Gaffky's Method for sections of 

 tissues: Harden the tissue in alcohol, place sec-i 

 tions for 20 to 24 hours in a deep-blue solution made 

 by adding a saturated alcoholic solution of methylene- ' 

 blue to distilled water, wash in distilled water, de- 

 hydrate in absolute alcohol, and clear in turpentine 

 and balsam. The best stains for the typhoid-bacillus 

 are Lceffler's methylene-blue and Ziehl's carbol-fuchsin. ; 

 The sections must remain in the dye at least 12 hours. ; 

 Baumgarten recommends Ehrlich's stain for tubercle- 

 bacilli. In this case a very dilute solution of acetic 

 acid is used for rinsing. The flagella may be shown by : 

 Lceffler's method. By leaving the specimens for several 

 days in Ehrlich's anilin-water fuchsin the spores may 

 be stained. Spores also appear if the stain is heated 

 and then extracted by nitric acid. Counterstain! 

 with methylene-blue. Coccidium oviforme. Gibbes'\ 

 Method. Sections of rabbit's liver, hardened in alco ' 

 hoi, are stained in a filtered solution of rosanilin sul 

 phate, 2 gm., in loo c.c. of a solution of anilin-oi'i 

 2 c.c, cologne 10 c.c, distilled water 88 c.c Ter; 

 minutes are required to stain. After washing it 

 water and then in alcohol, the sections are differen 

 tiated in a solution of iodin-green, 1 gm. in 100 c.c of. 

 the foregoing anilin-oil solution. When the red color hi;' 

 changed to a dull-purple, the substitution has pro 

 far enough. It is well to make an examination h 

 distilled water from time to time. Flagella. Tin; 

 first successful method was that of Koch, who used : 

 concentrated aqueous solution of hematoxylin, :'ti< 

 then transferred the cover-glass to a 5 per cent, sduj 

 tion of chromic acid or to Mailer's fluid. The flagelli 

 were stained brown-black. 1. Crookshank stains witll 



