STAINS 



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STAINS 



up the stain : Titrate alf f aqueous solution of eosin 

 against the solution of polychrome methylene-blue 

 until a precipitate just appears ; I c.c. of the poly- 

 chrome methylene-blue usually requires from 0.3 to 

 0.6 eosin solution. Then dilute both solutions to 

 make 0.02 fo solutions. The best violet stain is 

 obtained when a quantity of eosin is added to the 

 methylene-blue equal to one-half that required to pro- 

 duce a precipitate. This, however, must be deter- 

 mined for each individual solution. Staining : Blood 

 may be stained on either cover-slips or slides. 

 Cover-slips: Place blood films in a watch-crystal 

 with the stain. Heat until the fluid steams, and a 

 metallic scum forms on its surface. This requires 1 

 to 1*2 minutes. Alternately heat and cool for 6 

 minutes. Wash in water. Dry. Slides : Cover the 

 blood-film with the stain. Heat for 2 minutes. 

 Cool for 2 minutes. Heat again for 2 minutes. Wash 

 in water. Dry. Ruprecht's Method for the dem- 

 onstration of canaliculi of bone : File a section of 

 bone, thoroughly deprived of oil, to 0.3 mm. Place it 

 in ether for a minute, heat it quickly on a glass plate, 

 and while still hot return it to the ether. Transfer 

 to hot saturated alcoholic solution of " diamant " 

 fuchsin and cook for 5 minutes. Cool to below 34 C. 

 and then evaporate to dryness, at 70 . Scrape off the 

 superfluous dye and file between ground-glass plates, 

 with powdered pumice kept moist with a mixture of 

 benzine and vaselin (lO:l). Smooth on the whet- 

 stone, in the same mixture, with the fingers. Wash in 

 benzine, dry, and polish with writing-paper. Mount 

 in colophonium dissolved in warm, water-free ben- 

 zol. Sacharoff ' s Method for blood. Solution A. — 

 Saturated aqueous solution of methylene-blue di- 

 luted one-half with water. Solution B. — A I % aque- 

 ous solution of eosin (Griibler, w. g.). To solu- 

 tion A add solution B, stirring until a granular pre- 

 cipitate begins to form. (If no precipitate forms 

 the methylene-blue employed is not suitable for this 

 stain. ) After the precipitate begins to form solu- 

 tion B is added drop by drop. After each drop 

 a blood-film is covered with a portion of the mix- 

 ture, and the series of films so obtained is placed in 

 a moist chamber and allowed to stain for 24 hours. 

 Of these usually one or two will be found to be 

 good. The films are fixed " according to Ehrlich " by 

 heat. Sadowsky's Method. See Jacottet-Sadaivsky 

 Method. Saint-Remy's Method for the eggs of 

 tapeworms : Expel the eggs from the worm by com- 

 pression or laceration, from the last proglottid forward 

 as far as they can be found to secure successive stages 

 of development and arrange in sequence on slides. 

 Fix with Carnoy's fluid stain in alum carmin or 

 toluidin blue, and mount in toto in balsam. Salge- 

 Stoltzer Method for the study of rachitic bone : 

 Place the sections for 3 minutes in a 0.5% solution of 

 silver nitrate, wash in distilled water, place for one 

 minute in a 5% solution of sodium bromid, again 

 wash in distilled water, and develop in a neutral solu- 

 tion of amidol. The sections may be counterstained 

 with lithium carmin. Sand's Method for protozoa : 

 Fix in 2% osmic acid, wash in water, containing a 

 trace of ammonia, and mount in a drop of the follow- 

 ing solution : methylene-green 0.5 gm., glacial acetic 

 acid 2 c.c, glycerin 10 c.c, alcohol (9.4 ) 10 c.c, 

 distilled water 80 c.c Make up the loss by evapora- 

 tion with a drop of 10 <£ glycerin. Sayce's Medium 

 for the preservation of Crustacea: Glycerol 375 c.c, 

 90 alcohol 250 c c , water 250 c.c, corrosive subli- 

 mate 05 gm. Scarlet R. (Ger. Scharlaeh R ). one 

 of the azo-bodies which possess no salt-combining 

 group and which are characterized by their selective 



staining of fat. Cf. Miehaelis' Method. Schaffner's 

 Method. 1. For the artificial production of the sickle 

 stage of the nucleolus : Treat root-tips of the onion in 

 a mixture of absolute alcohol 95 c.c, chloroform 5 

 c.c, glacial acetic acid 1 c.c, 1% aqueous solution of 

 chromic acid I c.c. The cells of the peripheral layers, 

 where the action of the medium is most violent, show 

 the distortion of the nucleolus. 2. For the study of 

 mitosis : Fix root tips in chromic-acetic acid and stain 

 the sections first in anilin-safranin and then in picro- 

 nigrosin. 3. For making permanent mounts of pollen : 

 Spread a drop of albumen fixative on a slide, on this 

 sprinkle the fresh pollen, and stain with safranin and 

 gentian-violet (O. I gm. of each to 100 c.c. of absolute 

 alcohol). After 5 minutes clear in xylol and mount 

 in balsam. 4. A permanent stain for starch : Stain 

 for from 2 to 4 hours in a mixture of equal parts of 

 anilin water and saturated solution of safranin in 95^0 

 alcohol and for from 2 to 8 minutes in 2^ aqueous 

 solution of gentian-violet. Paraffin sections of the 

 young corms of Eiythronium give particularly favorable 

 results. Schardinger's Medium for the cultivation 

 of protozoa : Boil 30 or 40 gm. of hay or straw in 

 one liter of water; filter and add I <j/ t or 1.5 r c of agar- 

 agar ; cook until the latter dissolves, add sodium car- 

 bonate until the reaction is alkaline to litmus and, 

 without filtering, fill into test-tubes. Cultivate the 

 suspected material in this medium and isolate by the 

 method of dilution. Schmidt's Test for bilirubin : 

 Triturate particles of fresh feces in a saturated aqueous 

 solution of mercuric chlorid and let the suspension 

 settle for 24 hours. Bilirubin, if present, is colored 

 green and may be detected microscopically, when the 

 quantity is too small to be seen by the unaided eye. 

 Schmorl's Method for demonstrating the lacuna; 

 and canaliculi of bone : I . Fix in any but the subli- 

 mate solutions, preferably in the liquid of Midler or 

 Orth. Decalcify by any method, preferably a slow 

 one, as that of Ebner or Thoma, or in Muller's fluid 

 containing 3 J^ of nitric acid. Embed in celloidin. 

 Treat the section with water for IO minutes and stain 

 for from 5 to 10 minutes in thionin or in Nicolle's 

 carbol- thionin (see Nicolle's Method); wash in water 

 and treat for a minute with aqueous solution of picric 

 acid 1 saturated by heat and filtered when cold) ; wash 

 in water, then for 5 or IO minutes in jOfi alcohol ; 

 dehydrate in 95 ft alcohol and clear in oil of origanum. 

 Hematoxylin may be used prior to the picric acid, to 

 bring out the nuclei. The addition of a drop or two 

 of ammonia to the thionin will cause the canaliculi in 

 osteoid tissue to stain. Result: osseous matrix, yellow to 

 yellow-brown ; canaliculi and lacunse. brown to black ; 

 cells, red ; fat-cells (after fixation in Muller's fluid) 

 reddish-violet. 2. For immature bone : Fix very thin 

 pieces in M tiller' fluid or in Orth's followed by 

 Muller's, for from 6 to 8 weeks at room-temperature 

 or for 3 or 4 days in the thermostat. Wash in water 

 and decalcify after v. Ebner. Wash thoroughly, 

 harden in alcohol, and embed in celloidin. Stain very 

 thin sections for 3 minutes in ammoniated thionin, 

 and treat for a few seconds with saturated aqueous 

 solution of phosphotungstic or phosphomolybdic acid ; 

 wash in water for 5 minutes, or until the sections turn 

 sky-blue, and treat for from 3 to 5 minutes with dilute 

 ammonia 1 1 : 10). Dehydrate in alcohol, clear in 

 carbol-xylol. and mount in balsam. Overstaining may 

 be corrected by a few minutes' treatment with acid 

 alcohol, followed by washing in water, before de- 

 hydrating. Result : matrix clear to greenish-blue, 

 cells diffuse blue, borders of lacunae and canaliculi 

 bluish-black. In rachitic bone the canahculi are brought 

 out only in the osseous tissue. Schottelius' Method 



