STAINS, ETC. 



1404 



STAINING REAGENTS 



equal volumes of a and b. Sections must remain in 

 the mixture for 24 hours, and then be placed for y z an 

 hour in a saturated solution of oxalic acid. Methyl- 

 green and Bismarck Brown. Stain in Weigert's 

 Bismarck brown for a few minutes, wash, and stain in a 

 0.5 percent, aqueous solution of methyl -green. Clear 

 with bergamot-oil or xylol {List). Methyl-green 

 and Rosanilin Nitrate. Stain for a few minutes in a 

 0.5 per cent, aqueous solution of methyl-green, wash, 

 and stain for from 10 to 15 minutes in a 0.0001 per cent, 

 aqueous solution of rosanilin nitrate, and wash out 

 rapidly in absolute alcohol. Methyl-green and 

 Eosin. Mix 60 parts of methyl -green and I of eosin, 

 and dissolve in warm 30 per cent, alcohol. Sections stain 

 in from 5 to 10 minutes, and should be quickly washed in 

 successive alcohols (Calberla). Stain for a few minutes 

 in a mixture of 3 parts of absolute alcohol and 1 part 

 of a 0.5 per cent, aqueous solution of eosin, wash, and 

 stain for 5 minutes in a 0.5 per cent, aqueous solution 

 of methyl-green [List). Methyl-violet and Eosin. 

 [Schiejferdecker). Proceed as for anilin green and 

 eosin, using a 1 per cent, aqueous solution of methyl- 

 violet. Orcein. A vegetable dye obtained from tinc- 

 torial lichens, which unites in itself the properties of 

 basic and acid stains, and also 2 contrast-colors. A 

 saturated acetic-acid solution is used. After staining, 

 the section is washed in distilled water, immersed in 

 alcohol, and quickly transferred to cedar-oil, in which 

 it is mounted. Nuclei are colored blue and protoplasm 

 red. Pianese Double Stain. Prepare a saturated 

 solution of nigrosin in a saturated alcoholic solution of 

 picric acid ; mix 2 volumes of this with I volume of 

 anilin-water, and evaporate in open air. The crystals 

 deposited are dissolved in absolute alcohol, and from 

 this solution green crystals are obtained soluble in 

 alcohol, ether, and water. For tissues, make a 2 per 

 cent, solution in alcohol, for microorganisms, in water. 

 Stain sections first in lithium-carmin, treat with acid 

 alcohol, wash, and immerse in an alcoholic solution of 

 picro-nigrosin until they assume a brown hue. Decolor- 

 ize in oxalic acid. Nuclei are stained red, plasma dark- 

 yellow ; cartilage yellow ; connective tissue pale-green ; 

 elastic fibers violet. Picro-lithium-carmin. See 

 Orth's carmin stains. Ranvier's Ammonium-picro- 

 carmin. Dissolve I gm. of carmin in 3 c.c. of stronger 

 ammonia and 5 c.c. of distilled water by gentle heat, 

 then add 200 c.c. of a saturated, aqueous solution of 

 picric acid, boil, and filter. This stain is valuable for 

 complex tissues, like skin. Ranvier's Picrocarmin. 

 This gives a double stain without the use of an acid or 

 an alkali . Add a saturated , ammoniacal solution of car- 

 min to a saturated, aqueous solution of picric acid until 

 precipitation just appears, mix well, and leave protected 

 from dust, in shallow vessels, to crystallize. When the 

 bulk is reduced to l /$, decant the liquid, filter, and evap- 

 orate it to dryness on a water-bath. Dissolve the residue 

 and the crystalline deposit in water, filter, and evaporate 

 to dryness. A I per cent, solution of the resulting brown 

 powder yields a good stain. Renaut's Eosin-hema- 

 toxylin. Concentrated aqueous solution of potassium 

 eosin30c.c, saturated alcoholic solution of hematoxylin 

 40 c.c, saturated solution of potassium-alum in glycerin 

 (sp. gr. 1.26) 130 c.c. Mix, and stand aside for 5 or 6 

 weeks in a vessel covered with a sheet of perforated 

 paper, until the alcohol is evaporated, and then filter. 

 This reagent stains slowly. Objects may be mounted 

 in the solution diluted with from 1 to 2 volumes of 

 glycerin ; the tissues gradually absorb the colors, leaving 

 the medium colorless. This solution has a selective 

 action on the cells of salivary and gastric glands; 

 mucous cells become pale-blue, salivary ferment-cells 

 rose-colored. Safranin and Indigo-carmin (A'ossiits- 



ki). Sections are stained for from IO to 20 minutes 

 in a saturated solution of indigo-carmin, and, after wash- 

 ing in water and then alcohol, are stained in a 0.5 per 

 cent, solution of safranin in dilute alcohol. The same 

 author also recommends safranin and nigrosin, and 

 stains first in a o. I per cent, aqueous solution of nigrosin 

 for 3 to 5 minutes. Stohr's Picro-carmin. Dissolve 

 1 gm. of carmin in 50 c.c. of water and 5 c.c. of liquor 

 ammonia?, then add 50 c.c. of a saturated solution of 

 picric acid. Leave the mixture for 2 or 3 days in a 

 large, flat evaporating dish, then filter, and add a drop 

 of chloroform to the filtrate to preserve it. 'Weigert's 

 Picrocarmin. In a vessel protected from evaporation 

 soak for 24 hours 2 gm. of carmin in 4 gm. of ammonia. 

 Add 200 gm. of a concentrated solution of picric acid, 

 and put the whole away for 24 hours more. Acetic acid 

 is then added until the first precipitate appears. After 

 another 24 hours, a precipitate will be formed that can 

 only be partially removed by filtration. Now add 

 ammonia, drop by drop, at intervals of 24 hours, until 

 the solution becomes clear. If it stains too yellow, add 

 acetic acid ; if too red, a little ammonia. VII. Stains 

 for Microorganisms. Balmer-Franzel's Anilin 

 Gentian-violet. Gentian-violet 2 gm., anilin-water 

 iooc.c. Filter before using. Borofuchsin. This con- 

 sists of equal parts of boric acid and fuchsin dissolved in 

 dilute alcohol. Carbolic Black-brown. Black-brown 



1 gm., absolute alcohol 10 c.c, aqueous solution of car- 

 bolic acid 100 c.c. Dissolve the dye in the alcohol, and 

 add the carbolic acid. Cose and Simon's Gentian- 

 violet. Gentian-violet 2 gm.,ooper cent, alcohol 5 c.c, 

 anilin-water 100 c.c. Ehrlich's Anilin Gentian- 

 violet. Five c.c. of a saturated alcoholic solution of 

 gentian-violet and iooc.c. of anilin-water. Ehrlich- 

 Weigert's Solutions. 1 . Anilin-water 100 c.c. , con- 

 centrated alcoholic solution of fuchsin II c.c, absolute 

 alcohol 10 c.c. Methyl-violet may be substituted for 

 the fuchsin. Keeps about 2 weeks. 2. Saturated alco- 

 holic solution of methyl- violet 11 c.c, anilin-water 100 

 c.c, absolute alcohol 10 c.c. Fraenkel's Polychro- 

 matic Stain, a. Fuchsin-solution. b. Ninety per 

 cent, alcohol 50 c.c, distilled water 30 c.c, nitric acid 

 20 c.c, methylene-blue as much as will dissolve ; filter. 

 c. Alcohol 70 c.c, nitric acid 30 c.c, Bismarck brown 

 as much as will dissolve. Stain tubercle-bacilli prepar- 

 ations in solution a, then in b. The bacilli will appear 

 red and the nuclei and other bacteria blue. If a blue 

 stain is used first the specimen may be counterstained 

 in solution c, the nuclei then being brown. Fried- 

 lander's Fuchsin. Fuchsin I gm., glacial acetic acid 



2 c c , 90 per cent, alcohol 5 c.c, distilled water iooc.c. 

 Friedlander's Gentian-violet. See Staining of 

 Microorganisms, Pneitmo-bacilhts of Friea 

 Fuchsin. {Concentrated Alcoholic.) Fuchsin 25 gm., 

 absolute alcohol iooc.c. [Aqueous.) Fuchsin 1 gm., 

 90 per cent, alcohol 20 c.c, distilled water 80 c.c 

 Gentian-violet. I. [Concentrated Alcoholic.) 

 tian-violet 25 gm., absolute alcohol iooc.c. 2. (At/its- 

 ous.) Gentian-violet I gm. , 90 per cent, alcohol 

 distilled water 80 c.c. Gibbes' Double Stain. (See 

 Staining of . Microorganisms, Gibbes 1 Mr/hod. ) Gibbes 

 Magenta. Fuchsin (magenta) 2 gm. , anilin-oil > gm., 

 90 per cent, alcohol 20 c.c, distilled water 



Gold Orange. See Staining of Microorganisms, 

 L r nna's Method. Koch's Differentiating or Poly- 

 chromatic Stain, a. Koch's methylene -1 >lue solution. 

 b. Aqueous solution of Bismarck brown. Preparation! 

 of tubercle-bacilli are stained first in <;, then in />. '1 he 

 blue of the nuclei and other bacteria is changed I" 

 brown, while the tubercle bacilli retain the blue color 

 Koch's Methylene-blue. Saturated alcoholic methy- 

 lene blue solution 1 c.c, 10 per cent, solution of causae 



