STAINS 



511 



STAINS 



an aqueous suspension of a 24-hour culture of bac- 

 teria ; dry in air, fix in flame, and treat with a mordant 

 for 20 minutes ; wash, and treat with ammoniated so- 

 lution of silver oxid heated uutil it steams ; wash, and 

 treat for a few seconds with I % mercuric chlorid solu- 

 tion ; wash, and treat with the silver solution until the 

 film is light brown ; wash, and develop for a few sec- 

 onds in methyl alcohol, v. Wellheim's Stain. Mor- 

 dant the sections for from 6 to 1 1 hours in a very dilute 

 solution of ferric chlorid in 50^ alcohol, wash in 50',- 

 alcohol, and stain for several hours in a weak solution 

 of carminic acid in 50% alcohol. Whitney's Fluid 

 for fixing blood for differential staining : This is a 

 modification of Zenker's fluid, in which nitric acid is 

 substituted for acetic acid. Willcox's Method for 

 making permanent mounts of amebas : Put a drop of 

 water containing amebas on a cover-glass and carefully 

 absorb the excess of water with filter-paper. Fix 

 with a drop of picric alcohol, wash with 50^/ alcohol, 

 and dehydrate with absolute amyl alcohol. Attach to 

 the cover-glass by Overton's method, and stain. In 

 mounting, if supports are required, use strips of paper 

 soaked in xylol. Willebrand's Stain for double 

 staining the blood: Mix 25 c. c. each of concentrated 

 aqueous solution of methylene blue and 0. 5f« alco- 

 holic solution of eosin and add from 10 to 15 drops of 

 1% acetic acid. Winternitz's Method for tubercle 

 bacilli: Stain films or sections in 2f c solution of 

 fuchsin in anilin water. Decolorize in 5°fr alcoholic 

 solution of fluorescein to a light rose color. Counter- 

 stain in methylene-blue. Wolkowitsch's Method. 

 a. For bacilli of rhinoscleroma in sections: Stain for 

 from 24 to 48 hours in anilin- water gentian-violet; 

 wash, and treat for 3 or 4 minutes with iodin-potas- 

 sium iodid solution or for a few seconds with aqueous- 

 alcoholic solution of picric acid. Dehydrate in alco- 

 hol and clear in clove oil. The capsules stain best in 

 sections of alcohol material, b. For cover-glass films 

 of cultures of the bacilli of rhinoscleroma : Dry and 

 treat for a few seconds with acetic acid ; dry and stain 

 one minute in strong anilin-gentian- violet; rinse and 

 stain for a few seconds in 1 or 2£ eosin solution; 

 transfer to 60^ alcohol; wash in water; dry and 

 mount in balsam. Wood worth's Method for 

 graphic reconstruction of embryos : Draw an axial 

 line the length of the object, multiply by the magnifi- 

 cation. With a micrometer take the greatest diameter 

 of each section and plot them down transversely to the 

 axial line, at distances equivalent to the thickness of 

 the section multiplied by the magnification. Connect 

 the extremities of these diameters and thus obtain an 

 outline of the object. Measure on each section the 

 nearest and farthest boundary of the organs to be rep- 

 resented, plot them on the transverse lines and connect 

 the points, from section to section, and thus obtain the 

 outline of the organs. Wright's Stain for blood. 

 Preparation of the neutral stain. Solution A. — Make 

 a 0.5^ aqueous solution of the NaHCOj, being 

 careful to bring all of the salt into solution before 

 going on to the next step. Then add if( of methyl- 

 ene-blue (Griibler's methylene-blue, " Bx." "Koch," 

 or "Ehrlich's Rectified "l. Steam this in an Arnold 

 sterilizer for 1 hour after steam is up. Cool. Solu- 

 tion B. — o. I'r aqueous solution of eosin (Griibler, 

 "yellowish, soluble in water"). Add solution B to 

 solution A until the mixture becomes purple, a metal- 

 lic scum forms on the surface, and a finely granular 

 black precipitate appears in suspension. (About 5:0 

 c.c. of solution B to 100 c.c. of solution A.) Filter 

 off the precipitate. Do not wash it. Dry. Prepara- 

 tion of the staining solution. Make a saturated solu- 

 tion of the precipitate in pure methyl alcohol (0.3 gm. 



in 100 c.c. methyl alcohol). Filter, and add an addi- 

 tional 25 V of the original volume of methyl alcohol 

 used. This prevents precipitation of the stain on the 

 film. Cover the film with the stain for 1 minute. 

 Without pouring off the stain, add water drop by drop 

 until the mixture is translucent at the edges, and a 

 yellowish metallic scum forms on the surface. Stain 

 in this diluted stain for 2 to 3 minutes. Wash in 

 distilled water until the film becomes pink. Dry 

 between filter-papers. Staining reactions: Lympho- 

 cytes, nuclei dark purplish-blue; cytoplasm, robin's 

 egg blue. Large mononuclears, nuclei blue ; cyto- 

 plasm pale blue. Polymorphonuclear neutrophils, nu- 

 clei blue ; granules reddish-lilac. Eosinophils, nuclei 

 blue; granules blue. Mastzellen, nuclei blue to pur- 

 plish ; granules dark blue or purple. Myelocytes, 

 nuclei dark blue or lilac ; granules dark or reddish- 

 lilac. Blood-platelets stain blue or purplish. Ma- 

 larial parasites, nuclei, chromatin portion, lilac-red to 

 a black ; cytoplasm blue. Wiedemann's Method 

 for embedding the eyeball: Harden in 5<£ for- 

 malin, freeze in ice and salt, divide, and place for 2 

 days in glycerol and water. Dissolve I oz. of gelatin 

 in 8 oz. of water, add the shells and whites of 2 eggs, 

 filter, and add an equal volume of glycerol to the fil- 

 trate. Embed the eye in this mass and harden by ex- 

 posure to the vapor of formalin. Yamagiwa's 

 Method for neuroglia: Fix in Miiller*s fluid and 

 without washing harden for a week in absolute alco- 

 hol renewed daily. Embed in celloidin and stain the 

 sections for 12 hours in a saturated alcoholic solution 

 of eosin, for from 4 to 5 hours in a saturated aqueous 

 solution of anilin blue and differentiate in alkali- 

 alcohol (see Strobe' 's Method ') ; distilled water, dilute 

 alcohol, absolute alcohol, origanum oil, balsam. Axis- 

 cylinders deep blue, connective-tissue fibers pale blue 

 to green, glia cells black-violet, glia fibers red. 

 Yasuda's Medium for the culture of infusoria: Mix 

 I gm. of meat extract, 20 gm. of cane-sugar, 250 c.c. 

 of cooked concentrated infusion of Porphyra i-ulgaris, 

 and 729 c.c. of distilled water; sterilize, and introduce 

 the infusoria by means of a capillary tube. A pure 

 culture may be obtained by examining the tube under 

 the microscrope and emptying only that part of it con- 

 taining the desired species. Yersin's Medium for 

 the culture of plague bacilli. A mixture of a 2<V al- 

 kalized solution of peptone and a 2ft solution of gel- 

 atin. Zachariades' Method for the demonstration 

 of the ramifying bone-cells and their membrane: 

 Decalcify by picric acid; wash out all the acid. Treat 

 the sections for a few seconds with I ft osmic acid; 

 stain for 24 hours in weak aqueous solution of quino- 

 lein blue or for a few minutes in saturated solution of 

 safranin ; treat with a drop of 40*^ solution of caustic 

 potash warmed over a flame until they flatten. After 

 washing in water the sections may be mounted in 

 glycerol. Zacharias' Fluid. Glacial acetic acid I 

 part, absolute alcohol 4 parts, osmic acid a few drops. 

 An excellent medium for fixing kinetic nuclei and the 

 central nervous tissue. Wash in alcohol. Zalew- 

 ski's Method. 1. Harden the tissue for 24 hours 

 each in 65% alcohol and 95 fr alcohol, adding to the 

 latter a few drops of a strong solution of ammonium 

 sulfate and shaking it from time to time ; complete the 

 hardening in absolute alcohol with a few drops of am- 

 monium sulfate. The alcohol must fill the vessel to 

 the brim ; cork stoppers must not be used. 2. Harden 

 the tissue for 24 hours in 65 ft alcohol ; transfer into 

 I ft solution of potassium ferrocyanid in 95 ft alcohol ; 

 after 2 or 3 days transfer to a I ft solution of potassium 

 ferrocyanid in 65 ft alcohol ; place in 95 % alcohol 

 containing ift or 2f? of hydrochloric acid. After- 



