STAINS, ETC. 



1391 



STAINING OF MICROORGANISMS 



a drop of a saturated solution of gentian-violet in abso- 

 lute alcohol. Before evaporation takes place rinse 

 with water, dry, and mount in balsam. 2. Laffler s 



tod {Sternberg). A small quantity of a pure 

 culture is suspended in a few drops of distilled water. 



II drops of water are distributed upon clean glass 

 covers by means of a platinum-wire loop, and are sowed 

 with bacilli from the first drop. The little drops are 

 1 out by the platinum wire, allowed to dry in the 

 air. then passed through the flame in the usual way. 

 Care must he taken not to overheat. Lceffler's mordant 

 (No. I ) is placed upon the cover-glass so as to fully cover 



.in arched drop. The cover-glass is then heated 

 until steam begins to arise ; too much heat produces 

 a precipitate. The mordant is left for ]/ 2 to I minute 

 upon the cover-glass and gently moved about. Wash 

 in distilled water, then in absolute alcohol to remove 

 all remnants of the mordant. Drop the staining solu- 

 tion (preferably neutral saturated anilin-water fuch- 



so as to fully cover the glass, and heat till vapor 

 begins to be given off"; wash, dry, mount. 3. 

 Trenkmann's Method (Schenh). A few drops of 

 boiled water are placed upon a slide, and a small drop 

 of the culture added and intermixed. Of this mixture 

 a droplet is spread upon a cover-glass, dried in air, 

 laid, without heating, in a 2 per cent, solution of 

 tannin and 0.5 per cent, hydrochloric acid, in which it 

 is allowed to remain for from 6 to 12 hours. It is then 

 washed in water and placed in iodin-water for half an 

 hour, washed, and carried to a weak solution of anilin- 

 oil gentian-violet for half an hour, again washed, then 

 dried, and mounted. Gonococcus. The gonococci 

 are seen in the pus-cells grouped around the nucleus. 

 Watery solutions of anilin dyes, preferably methylene- 

 blue, stain the cocci intensely. Gram's method decol- 

 orizes them. 1. Sc/iutz stains for 5 or 10 minutes in a 

 saturated solution of methylene-blue in 5 per cent, car- 

 bolic-acid water and differentiates for 3 seconds in 

 acetic acid 5 parts, water 20 parts, washes in water, 

 and counterstains in a dilute solution of safranin. 

 nzynski's solution of methylene-blue and eosin 

 stains the cocci blue and the pus-cells pink. 3. 

 r stains for 2 or 3 minutes in a concentrated alco- 

 holic solution of eosin, slightly warmed ; removes the 

 excess with filter-paper, and counterstains in a concen- 

 trated alcoholic methylene-blue solution for from a quarter 

 to half a minute. The cocci appear blue, the cells red. 

 Influenza-bacillus ( of Pfeiffer and Canon). 

 Canon s Method of staining it in blood : Spread the 

 blood upon cover-glasses in the usual way. When 

 dry, place the covers in absolute alcohol for 5 minutes 

 and then transfer them to Czenzynski's staining solu- 

 tion. In this they remain for from 3 to 6 hours in 

 an incubating oven at 37 C, when they are washed, 

 dried, and mounted. The bacillus also stains well in 

 Lceffler's methylene-blue, with heat, and in dilute 

 Ziehl's solution. It does not stain by Gram's method. 

 Koch's Comma-bacillus. Stains easily in diluted 

 alcoholic or concentrated solutions of anilin dyes ; if 

 cold, in about 10 minutes ; if heated, more quickly 

 and more deeply. Gram's method is not applicable. For 

 sections use fuchsin or methylene-blue. I. Koch's 

 Method. Stain sections well hardened in absolute 

 alcohol for 24 hours in strong aqueous solution of 

 methylene-blue. If the staining fluid be warmed, 

 less time is necessary (Crookshank). 2. Nicati and 

 ■h's Method ( Crcokshank ). A small quantity of 



stools or of the scrapings of the intestinal mucosa is 



spread and dried upon a slide, steeped for a few seconds 



m corrosive sublimate solution or in I per cent, osmic 



acid, and stained in fuchsin-anilin solution. 3. Babes' 



>d. Place sections for 24 hours in aqueous solution 



of fuchsin ; wash in distilled water faintly acidulated with 

 acetic acid or in I per cent, corrosive-sublimate solution ; 

 carry them quickly through alcohol and oil of .cloves. 

 Phagocytes. Sections of a Peyer's patch, hardened 

 in absolute alcohol and embedded in paraffin, are 

 stained first with alum-carmin, and then with gentian- 

 violet by Gram's method. The bacilli are stained a 

 deep bluish -violet. Plasmodium malariae. A 

 minute drop of blood from the finger-end is spread 

 upon cover-glasses and the film fixed by heat in the 

 usual way, or by placing the covers in a mixture ol 

 equal parts of absolute alcohol and ether. Czenzyns- 

 ki's solution may be used to stain, the red cor- 

 puscles taking up the eosin, the malarial organisms the 

 methylene-blue. Plehn recommends methylene-blue, 

 concentrated aqueous solution 60 parts, eosin 0.5 per 

 cent, solution in 75 per cent, alcohol 20 parts, 20 per 

 cent, potassium-hydroxid solution 12 parts. Stain for 

 5 or 6 minutes and wash in water. Pneumo-bacil- 

 lus of Friedlander. Friedlander^ s Method. Stain for 

 24 hours in warm, concentrated alcoholic solution of 

 gentian-violet 50 parts, distilled water loo parts, glacial 

 acetic acid 10 parts ; then wash in I per cent, acetic 

 acid, dehydrate in alcohol, dry or clear, and mount. 

 The capsule is pale-blue, the center deep-blue. For 

 cover-glass preparations Friedlander advises the follow- 

 ing : place in acetic acid for a few minutes ; displace 

 the acid by blowing upon the preparation through 

 a pipet; stain a few seconds in a saturated solution 

 of anilin-water gentian -violet ; rinse in water; dry with 

 filter-paper; mount in balsam. Pneumococcus of 

 Fraenkel, Klebs, Eberth, and Weichselbaum. 

 This may be stained by Gram's method. Methods to 

 demonstrate the capsules. I. Ribbert places the pre- 

 paration for a few minutes in a hot, saturated solution 

 of dahlia-violet in water 100 parts, alcohol 50 parts, 

 glacial acetic acid 12.5 parts. Staining takes place 

 rapidly ; wash quickly in water. The capsules appear 

 light-blue, the cocci dark-blue. An overstain makes 

 the capsules also dark -blue. 2. Roux stains for a few 

 seconds in an aqueous solution of methyl-violet, and, 

 without washing, counterstains for the same length of 

 time in Lceffler's alkaline methylene-blue. 3. C. v. Kahl- 

 den stains for some hours in a 1 per cent, solution of 

 fuchsin in 5 per cent, carbolic acid. The capsule appears 

 pale-red, the coccus deep-red. Protozoa in Carcin- 

 oma. Russell 's Method. Stain sections for 10 minutes in 

 a saturated solution of fuchsin in 20 per cent, carbolic 

 acid, wash in water, then in absolute alcohol, and stain 

 for 5 minutes in a I per cent, solution of iodin-green 

 in 2 per cent, carbolic acid; dehydrate in absolute 

 alcohol, clear in clove-oil, and mount in balsam. 

 The " parasites " are red or purple, the tissue-cells 

 blue. Spirochaetae obermeieri. Stain with Lceffler's 

 solution or any of the common basic anilin dyes. 

 Giinther's Method. Air-dried cover glasses are placed 

 in 5 per cent, acetic acid for 10 seconds, removed, and 

 the acid displaced by blowing upon the preparation 

 through a glass tube and then holding it for a moment, 

 film-surface downward, over strong ammonia just pre- 

 viously shaken. Then stain in gentian-violet anilin- 

 water or in an aqueous solution of other anilin dyes, and 

 proceed as usual. The acetic acid bleaches the red 

 blood-corpuscles Spores. Spores are more resistant 

 to the action of stains than other parts of bacteria. I . 

 Giinther's Method. Float the prepared cover-glass on 

 a solution of anilin-water fuchsin or gentian-violet 

 heated to boiling, and remove from the flame for a 

 minute. Repeat this process 5 times and then wash 

 in a 3 per cent, solution of acetic acid in alcohol, and 

 afterward in water. 2. Moller 1 s Method (Sternberg). 

 Dry the cover-glass preparation in air, pass it thrice 



