STAINS, ETC. 



1389 



STAINING OF MICROORGANISMS 



ehydrated, cleared, and mounted. 4. PlattV s Method. 

 Float sections for 10 minutes in a solution of magenta, 

 2 parts, anilin-oil 3 parts, alcohol (sp. gr. 0.830) 20 

 parts, distilled water 20 parts (Gibbes). Warm the 

 solution to 45 C. Rinse in water, and counterstain 5 

 to 10 minutes in concentrated alcoholic solution of 

 picric acid. Wash in water for 5 minutes and then in 

 alcohol for 15 minutes. Pass through absolute alcohol 

 and clove-oil, and mount. The clubs appear red, the 

 tissue yellow. Better than the magenta is the Ziehl- 

 Neelsen solution. When this is used, remove the picric 

 acid by long immersion in alcohol, and counterstain 

 with gentian-violet or methylene-blue (Crookshank). 

 Amoeba coli. Councilman and Lajleur 's Method. 

 Sections of tissue hardened in alcohol are stained in 

 Lceffler"s methylene-blue. The amebae are colored dark- 

 blue. The nuclei are best demonstrated by fixing in 

 Flemming's solution and staining with safranin, by 

 which method the nucleoli may often be seen {Schenk). 

 Cover-glass preparations may also be made. Bacillus 

 anthracis. Kuhne's Method. Place the section for 5 

 minutes in carbolic black-brown solution, rinse in 

 lithia -water and then in 90 per cent, alcohol, stain 5 

 minutes in carbol-fuchsin, and decolorize in fluorescein- 

 alcohol. The carbolic black-brown acts as a mordant 

 and fixes the red color. Bacillus of Eve and Lin- 

 gard. Stains easily by Gram's method and in ordinary 

 anilin dyes, but not by Lustgarten's method. Bacillus 

 leprae. Stains with the anilin dyes and by Gram's 

 method. Double-stained preparations are easily made. 

 I. Babes' Method. Stain in a solution of rosanilin 

 hydrochlorate in anilin-water ; decolorize in 33 per 

 cent, nitric acid. Use methylene-blue as a contrast- 

 stain for the nuclei. 2. Baumgarteri 's Method (C. 

 v. Kahlden). Stain 6 to 7 minutes in a dilute alcoholic 

 solution of fuchsin (5 drops of a concentrated solution 

 to a watch-glassful of water) ; decolorize ^ minute in 

 nitric acid I part, alcohol 10 parts ; wash in water, 

 counterstain in methylene-blue; wash again in water, 

 dry, and mount in balsam. Lepra-bacilli appear red 

 upon a blue ground. A longer time is necessary for 

 staining tubercle-bacilli. 3. Lustgarten's Method. 

 Cover-glass preparations are stained with anilin-water 

 fuchsin, or gentian-violet; decolorized by 1 per cent, 

 sodium hypochlorite ; rinsed in water ; dried; mounted. 

 Bacillus of Lustgarten. 1. Lustgarten's Method. 

 Place thin sections in Ehrlich-Weigert's gentian-violet 

 solution for 12 to 24 hours, or 2 hours at 40 C. Wash 

 thoroughly in alcohol ; transfer for 10 seconds to a 1.5 

 per cent, solution of potassium permanganate. A pre- 

 cipitate is formed that adheres to the section ; wash in 

 a dilute aqueous solution of pure sulphuric acid, then 

 in water, and if not decolorized return to the potassium- 

 permanganate solution and repeat the process. When 

 decolorized, dehydrate and mount in balsam. Cover- 

 glass preparations are washed off in water instead of in 

 alcohol. Tubercle- bacilli and lepra-bacilli may be 

 stained in the same way. 2. De GiacominV s Method. 

 Leave sections 24 hours in anilin-water-fuchsin ; wash in 

 water; decolorize in ferric chlorid, first in a dilute, then 

 in a saturated solution. Counterstain with Bismarck 

 brown. Stain cover-glass preparations for a few 

 minutes in the heated solution. Bacillus mallei. 

 This bacillus stains in aqueous solutions of anilin colors, 

 and best when the solution is feebly alkaline. I. Add 

 to 3 c.c. of a 1 : 10,000 solution of caustic potash I c.c. 

 of a saturated alcoholic solution of an anilin color ; or 

 the anilin-water-fuchsin or methyl-violet solution of 

 Ehrlich may be used, diluting it at the moment of using 

 with an equal quantity of a 1 : 10,000 solution of caustic 



JOtash {Sternberg). 2. Lceffler advises for cover-glass 

 preparations, heating for 5 minutes in Ehrlich's solu- 



tion, then decolorizing in I per cent, solution of acetic 

 acid to which tropeolin has been added to give it the 

 yellow color of Rhine wine, and washing quickly in 

 distilled water. 3. For sections, LcefHer advises his 

 alkaline methylene-blue ; decolorizes in a mixture of 

 distilled water 10 c.c, strong sulphuric acid 2 drops, 

 5 per cent, oxalic acid 1 drop. Sections are left in 

 this about 5 seconds. 4. A'oniewicz' s Method {Schenk). 

 Sections are transferred from alcohol to alkaline 

 methylene-blue for 2 to 5 minutes, rinsed in water, and 

 decolorized in a mixture of 0.5 per cent, acetic acid 75 

 parts and 0.5 per cent, aqueous solution of tropeolin 

 OQ 25 parts. Thin sections are only dipped quickly into 

 the solution ; thick sections remain 2 to 5 seconds. 

 W ash with water ; spread upon the slide, dry in air or 

 over a flame, clear in xylol, and mount in balsam. The 

 bacilli appear black on a blue ground. 5. Schiitz' ' s 

 Method. Sections or cover-glass preparations are placed 

 for several hours in an aqueous solution of methylene- 

 blue, then washed in weak acetic acid (0.5 c.c. to 100 

 c.c), dehydrated in alcohol, cleared in cedar-oil, and 

 mounted in balsam (after Squire). Bacillus tetani 

 may be stained by Gram's method. For double stain- 

 ing of bacilli and spores, the method of Ziehl may be em- 

 ployed. Bacillus tuberculosis. I. . -liens' Chloroform 

 Method (Sc/icnk). Three drops of absolute alcohol are 

 dropped in a watch-glass upon a crystal of fuchsin the 

 size of a millet-seed, and 2 to 3 c.c. of chloroform are 

 added. This solution becomes turbid and then clears 

 When clear, the cover glass preparation is laid in it for 

 from 4 to 6 minutes, until the chloroform is evaporated, 

 then decolorized in concentrated alcohol to which hydro- 

 chloric acid (3 drops to a watch-glassful) has been added, 

 rinsed in water, and counterstained with dilute methyl- 

 ene-blue. 2. CzaplewskVs Methods, (a) After staining in 

 warm Ziehl's solution, drain, and immerse 6 or 10 times 

 in as aturated alcoholic solution of fluorescein containing 

 an excess of methylene-blue. Counterstain in a saturated 

 solution of methylene-blue, and wash quickly in water. 

 (b) Stain the cover-giass preparation in carbol-fuchsin, 

 heating it until clouds of vapor arise. The following 

 solution is then dropped upon the slide, held ob- 

 liquely, until the surface is cleared : hydrochloric 

 acid and sodium chlorid each 2.5 parts, dissolved in 

 distilled water 100 parts ; then add 500 parts of alcohol. 

 The slide is then washed with water, dried in air, and 

 treated with a drop of some immersion-oil that can 

 be removed by xylol. (Arbeiten aits dem Path. 

 Anat. Institut zu Tubingen, 1892, Bd. I, Heft 

 3. Also Monatsckr. f. prakt. Derm. XVII, No. 4.) 

 3. Ehrlich's Method ( Schenk ) for tubercle-bacilli 

 in pus. Spread the pus very thinly; place the 

 cover-glass for I to 2 hours in cold anilin -fuchsin ; 

 decolorize with nitric acid I part, and sulphanil-nitric 

 acid, saturated solution, 3 to 6 parts. The after-stain 

 is methylene-blue. 4. FrwnkeVs Method (Schenk). 

 The cover-glass is stained with anilin-water-fuchsin, 

 transferred to a fluid consisting of a saturated solution 

 of methylene-blue in 50 parts of water, 30 of alcohol, 

 and 20 of nitric acid. When the preparation appears 

 blue, it is washed in alcohol and acetic acid or in pure 

 water. 5. Friedlander' s Method. Place upon the dried 

 film-surface of the cover-glass 3 or 4 drops of 

 carbol-fuchsin, heat until vapor arises, wash in dis- 

 tilled water, drain, and add a few drops of a decolor- 

 izing solution (pure nitric acid 5 c.c. to 80 per cent, 

 alcohol 100 c.c); decolorization takes place quickly. 

 Wash in water, counter-stain about 5 minutes with 

 methylene-blue, without heat: wash; dry and mount. 

 6. Gabbefs Modification of FrcenkeFs Method. Stain 

 in Ziehl's carbol-fuchsin for about 2 minutes, without 

 heating; wash in water; stain in 25 per cent, sul- 



