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STAINS 



hours in polychrome methylene-blue or thionin, and 

 after rinsing in water differentiate in glycerin-ether for 

 15 minutes; wash in water; alcohol, bergamot oil, 

 balsam. Cf. Unna' s Method. 2. For the granules 

 of mast-cells: Stain sections for 24 hours in fuchsin- 

 anilin-water, decolor to a light red in 50% alcoholic 

 solution of fluorescein, and afterstain in alcoholic solu- 

 tion of methylene-blue. Kostanecki-Siedlecki 

 Mixture : Equal volumes of saturated sublimate solu- 

 tion and 3^? nitric acid. Absolute alcohol in the same 

 proportion may be added. Fix for 24 hours and wash 

 in iodin alcohol. See Zenker's Fluid. Kukenthal's 

 Method for paraffin sections: Dissolve the dye in 

 absolute alcohol and add it dropwise to turpentine until 

 the desired color is produced. O verstaining is corrected 

 by treatment with a mixture of equal parts of alcohol 

 and turpentine. Use olive-oil collodion fixative. 

 Kultschitzky's Hematoxylin. I. Hematoxylin 

 (with enough absolute alcohol to dissolve it) I part, 

 saturated solution of boric acid 20 parts, distilled water 

 80 parts. Let the fluid stand for 2 or 3 weeks— until 

 the yellow color changes to red. Just before using add 

 to a watchglassful of the stain a few drops of acetic 

 acid. 2. Dissolve I gm. of hematoxylin in a little 

 alcohol and add 100 c.c. of 2f acetic acid. Kult- 

 schitzky's Method for neuroglia: Stain paraffin sec- 

 tions for from 5 to IO seconds in rulein S prepared as 

 follows: Rulein S 1 gm., 2$ acetic acid 400 c.c, 

 saturated solution of picric acid 400 c.c. Wash in 

 alcohol. Land's Method for mitosis in plant cells: 

 Fix the ovules in chromic-acetic acid for 2 hours at a 

 temperature of Ioo° C. Stain prefertilization stages in 

 Flemming's safranin-gentian- violet-orange-mixture or 

 in Heidenhain's iron-hematoxylin ; for the stage of 

 fertilization use cyanin and erythrosin, after treatment 

 with acetic a"cid and chloroform. Lang's Method 

 for gonococci : Stain the film for from 15 to 30 minutes 

 in a mixture of 4 volumes of saturated solution of thio- 

 nin and one volume of saturated solution of fuchsin in 

 2% carbolic acid. Langhan's Method for the 

 demonstration of glycogen in tissue cells : Harden 

 perfectly fresh tissue (e. g., the kidneys of a diabetic 

 subject) in absolute alcohol; stain inLugol's solution; 

 dehydrate in a mixture of tincture of iodin 1 part and 

 absolute alcohol 4 parts; clear and mount in oil of 

 origanum. Laurent's Stain : The exact proportional 

 relations of the eosin-methylene-blue mixture are ob- 

 tained by pouring together 1000 c.c. of 1 r / solution of 

 eosin and 882 c.c. of I ^ methylene-blue. After 48 

 hours the neutral pigment precipitates. Immediately 

 before using shake the mixture, add 4 volumes of water, 

 and boil. Then stain for from a half to 6 hours. 

 Transfer without washing to absolute alcohol ; xylol; 

 balsam. Lavdowsky's Fluid. I. Distilled water 

 20 parts, 95 <f c alcohol 10 parts, formalin 3 parts, glacial 

 acetic acid 0.5 part. 2. Distilled water 30 parts, 95% 

 alcohol 15 parts, formalin 5 parts, glacial acetic acid I 

 part. Lavdowsky's Method for staining nerve tissue 

 by immersion in methylene-blue : Mix the white of an 

 egg with an equal volume of 0.25% solution of 

 ammonium chlorid and in this dissolve from O.I', to 

 o.2'/e of methylene-blue. Immerse the tissue while it 

 is still warm. Laveran's Stain for blood : In a 150 

 c.c. flask dissolve "some" AgNO s in 5occ. or 60 c.c. 

 of water. When dissolved fill the flask with a solution 

 of NaOH (percentage not given). Wash the precipi- 

 tate of AgO with distilled water to remove the excess 

 of NaOH and the NaON 3 formed. Then add a sat- 

 urated aqueous solution of methylene-blue medicinale 

 (Hochst) and let the mixture stand for 7 or 8 days, 

 shaking it occasionally. Decant. The product so ob- 

 tained Laveran terms " bleu Borrel." To stain, Laveran 



mixes methylene-blue (bleu Borrel), I c.c; eosin 

 O. \ c /o aqueous solution, 4 c.c; distilled water, 6 c.c. 

 Stain 12 to 24 hours. Rinse in water. Wash in 5% 

 aqueous solution of tannin for I to 2 minutes. Wash 

 in water. Dry. Films are previously fixed in absolute 

 alcohol for 20 minutes. Lazear's Execution of 

 Nocht's Modification : Solution A. The polychrome 

 methylene-blue solution. To al/r aqueous solution 

 of methylene-blue add \f of NaOH. Heat in a 

 water-bath for several hours. Cool, then filter. To 

 neutralize this solution, add dilute acetic acid until blue 

 litmus paper is turned red above the line which the 

 methylene-blue stains. Then add more alkaline poly- 

 chrome methylene-blue until the solution just fails to 

 turn blue litmus red. To this neutralized solution add 

 an equal volume of distilled water ; then a saturated 

 solution of ordinary methylene-blue until the poly- 

 chrome methylene-blue has lost its red color — about I 

 part of the former to IO parts of the latter. Solution 

 B. A 0.2^ aqueous solution of eosin. With burets de- 

 termine the proportions of the two solutions, on mixing 

 which, a fine slack precipitate is obtained, and a scum 

 forms on the surface of the mixture. This may require 

 I part of Solution A to 3 parts of Solution B, or the 

 reverse. Once determined, the proportion remains 

 constant. Blood-films are fixed for. I to 2 minutes in a 

 0.25,% solution of formalin 11195% alcohol. To stain: 

 Remove scum from the surface of the stain with filter- 

 paper. Place the films face down in the stain. Stain 

 for 3 to 24 hours. Lee's Method: A "dry" cel- 

 loidin process. Infiltrate after Gilson or in the usual 

 way; embed in a paper tray and harden in vapor of 

 chloroform for from an hour to overnight; turn the 

 object from time to time. Clear in a mixture of equal 

 parts of chloroform and cedar oil ; add oil from time to 

 time and gradually convert the mixture tc nearly 

 pure cedar oil. When clear, expose the mass to air and 

 the chloroform will evaporate. Preserve in a stoppered 

 bottle. Cut with the block and the knife dry. Leish- 

 man's Stain for b'.ood: Preparation of the neutral 

 stain. Solution A. The solution of polychrome meth- 

 ylene-blue. A 1 </o aqueous solution of methylene-blue 

 med. (Grubler) is made alkaline with 0.5^ Na 2 CO :l . 

 This is heated for 12 hours at 65 C, and then allowed 

 to stand for 10 days before use. Solution B. A o. 1 ' , 

 aqueous solution of eosin (extra BA Grubler). Equal 

 parts of Solutions A and B are mixed in an open vessel 

 and allowed to stand for 5 or 6 hours, with occasional 

 stirring. The precipitate formed is collected on a filter, 

 washed with water, dried, and powdered. The stain 

 ing solution : Dissolve o. I gm. of the dry precipitate 

 in 100 c.c. pure methyl alcohol (Merck "foranalysi 

 To stain : four drops of the solution are poured on the 

 blood-fiun, and allowed to stain for V 2 minute. With 

 out pouring off the stain, 6 drops to 8 drops of distilled 

 water are added and the mixture is allowed to stain for 

 5 minutes. Wash gently. Put few drops of water on 

 the blood-film for I minute. Then dry, and mount. 

 Staining reactions: Red blood-corpuscles stain pale 

 pink or greenish. Lymphocytes — nuclei stain dark 

 ruby red ; protoplasm stains pale blue. Mononuclears 

 — nuclei stain ruby red; protoplasm stains pale blue. 

 Polymorphonuclear neutrophils — nuclei stain ruby red ; 

 granules stain red. "Coarse-grained eosinophils" — 

 nuclei stain ruby red; granules stain pale pink. Baso- 

 phils — nuclei stain red ; granules stain purplish black. 

 Blood-platelets stain deep ruby red. Malarial parasites 

 — nuclei, chromatin portion stains ruby red; cytoplasm 

 stainsblue. v. Lenhossek's Method for ganglion-cells: 

 I. Stain sections of formalin material for 5 minutes in 

 concentrated aqueous solution of thionin, wash in water, 

 differentiate in a mixture of anilin I part and absolute 



