STAINS 



506 



STAINS 



Then dry it. Preparation of the staining solution. 

 Dissolve the dry precipitate in hot absolute alcohol 

 (ethyl), using 0.2 gin. precipitate to loo c.c. alcohol. 

 Filter. Add 2 c.c. anilin oil to 100 c.c. staining solu- 

 tion. Of this solution add 1 or 2 drops to I c.c. dis- 

 tilled water (or 30 drops to 20 c.c. water). In this 

 mixture stain fresh films for 20 minutes to y 2 hour; 

 older films from 3 to 4 hours. Fix blood-films for 1 

 hour in a mixture of equal parts of absolute alcohol 

 and ether. Staining reactions : Red blood- corpuscles 

 stain pale orange. Malarial parasites — nuclei, chro- 

 matin stains violet ; cytoplasm stains blue. Rib- 

 bert's Method. See Grant's Method, Rieder's 

 Method for the selective staining of fat. See Sudan 

 III. Rindfleisch's Method for blood sections: 

 This is the same as Arnold's, except that after fixation 

 the blood is mixed with glycerol. Robertson's 

 Method for a " black reaction " in tissue elements of 

 the central nervous system : Place the object in a 

 large quantity of a I °/ solution of platinum chlorid 

 containing 5 % of formalin and so close the bottle as 

 not to exclude the air entirely. The reaction appears 

 in from I to 3 months and should be continued for 

 several weeks more, fresh platinum solution being 

 added if necessary. Transfer the tissue to a solution 

 of dextrin and cut on a freezing microtome. Dehy- 

 drate, clear, and mount in the usual way. Robin's 

 Method for preserving sputum for microscopic exami- 

 nation : Add to the sputum an equal volume of 5% 

 solution of carbolic acid, 5% formalin, or 2% trikresol, 

 and mix by shaking. Romano wsky's Stain for blood : 

 I. The concentrated stain. To a saturated aqueous 

 solution of methylene-blue I part, add a I c / aqueous 

 solution of eosin 2 parts. Mix these in a watch-crys- 

 tal, stirring with a glass rod. Prepare the stain only I 

 to 2 minutes before use. Float blood-films on the sur- 

 face of the stain. One-half to one hour is sufficient to 

 give the violet chromatin stain. Two to three hours 

 are necessary to give the most intense stain. 2. A 

 more dilute form of the stain. A saturated aqueous so- 

 lution of methylene-blue, distilled water, equal parts. 

 To this add an equal volume of 0.5% aqueous solu- 

 tion of eosin. Mix in watch-crystal as in I. Stain 

 for 24. hours. Wash in distilled water. Dry. Roman- 

 owsky used a solution of methylene-blue over the sur- 

 face of which mold had formed. Fixing of blood- 

 films : Heat for 30 minutes at 105 to Iio° C. Stain- 

 ing reactions : Red blood-corpuscles stain rose-red. 

 Leukocytes — nuclei, stain dark violet ; those of eosino- 

 phils a reddish-violet; eosinophil granules stain in- 

 tense red; neutrophil granules stain dark violet; pro- 

 toplasm lymphocytes stain dark blue ; mastzellen 

 stain dark blue. Blood-platelets stain dark, reddish- 

 violet. Malarial parasites — body stains blue ; center 

 of achromatic area stains carmin violet. Romanow- 

 sky-Ziemann's Stain: Prepare a 0.1% solution 

 of eosin and a I % solution of methylene-blue ; when 

 the latter is entirely dissolved mix the two solutions in 

 the proportion of 5 : I. Stain sections for a half- hour 

 and wash in a stream of water. Mount in xylol-balsam. 

 Rose de Naphthaline. The same as Magdala red. 

 Rosenberger's Method. 1. For staining blood: 

 Fix the films by heat or in absolute alcohol or alcohol 

 and ether and stain in a mixture of 10 c.c. of a satu- 

 rated aqueous solution of methylene-blue, 4 c.c. of a 

 saturated aqueous solution of phloxin, 6 c.c. of 95% 

 alcohol, and 12 c.c. of distilled water. 2. For stain- 

 ing the tubercle bacillus : The essential point in this 

 process is the use of sweet spirit of niter for bleaching; 

 it is also mixed with alcoholic solutions of methylene- 

 blue, malachite green, bismarck brown, and gentian- 

 violet. Rosin's Method. 1. For the central nervous 



system: Stain sections for 5 minutes in Rosin's 

 mixture, wash for 2 minutes in distilled water, and 

 transfer for 5 or 10 seconds into acetic acid 1 : 2000 ; 

 wash one minute in water, dehydrate in absolute 

 alcohol, clear in xylol, mount in balsam. Result: 

 colored blood-cells and medullary sheaths are orange 

 (only in chromium preparations) ; blood-vessel wails 

 and sclerosed neuroglia are purple ; axis-cylinders, 

 ganglion-cells, leukocytes, nuclei and nucleoli of some 

 ganglion-cells and cytoplasm of glia-cells are red ; 

 nuclei of glia-cells, bloodvessel walls, the connective 

 tissue and the leukocytes are blue-green. 2. For 

 ganglion-cells : Stain in saturated aqueous solution of 

 neutral red, wash in water and dehydrate in alcohol 

 that is free from acid. Granules of Nissl red, nucleoli 

 red, all else yellow. 3. For pigment in ganglion- 

 cells : Treat the tissue with formalin, cut on the 

 freezing microtome, place the sections for 24 hours in 

 a saturated solution of sudan III in 80 f c alcohol, and 

 mount in glycerol. Rosin's Stain : 1. Ehrlich's 

 triple-stain mixture 0.4 part, distilled water 100 parts, 

 0.5 acid fuchsin solution 7 parts. 2. Prepare con- 

 centrated aqueous solutions of acid eosin and basic 

 methylene-blue and mix them. The combination 

 produces a new dye, the eosinate of methylene-blue, 

 which stains acid substances blue, alkaline substances 

 red, and neutral substances violet. Nerve-cells are 

 an exception ; in them the cytoplasm takes the red, 

 the Nissl bodies the blue color, while the nuclei are 

 not blue. Rossolimow and Murawiew for nerve- 

 fibers : Harden in 2jJ formalin for 2 days, then in 

 4fo for 2 days; tease or section, and stain in heated 

 methylene-blue ; differentiate in anilin-alcohol after 

 Nissl and clear in cajeput oil. Rothberger Reac- 

 tion, a test for Bacterium coli commune. Add 3 or 

 4 drops of concentrated solution of neutral red to 10 

 c.c. of liquid agar and 0.5 c.c. of a 24-hour culture of 

 Bacterium coli. In about 24 hours the culture be- 

 comes strongly fluorescent. This reaction is said to 

 be specific. Rothig's Stain, (a) Dissolve 0.5 gm. 

 of kresofuchsin in 100 c.c. of 9$% alcohol and 3 c.c. 

 of hydrochloric acid, (b) Dilute a saturated solu- 

 tion of picric acid with 2 volumes of water. Mix 40 

 c.c. of a and 32 drops of /' and stain in this for from 

 2 to 24 hours ; wash in 95 c /c alcohol ; absolute alcohol, 

 xylol, balsam. Orange G may be used as a counter- 

 stain. Hematoxylin may be used to forestain. 

 Rothig used material fixed in sublimate solution. 

 Rousseau's Method. I. For decalcifying very deli- 

 cate objects : Embed fixed material in celloidin ; 

 treat it with 85$ alcohol; decalcify in a mixture 

 of from 15% to 40% of nitric acid in alcohol ; wash 

 in alcohol containing precipitated calcium carbonate, 

 and cut sections. 2. For decalcifying : Place an ob- 

 ject embedded in celloidin in a covered caoutchouc 

 dish containing a mixture of alcohol 50 c.c. and 20 to 

 30 drops of hydrofluoric acid ; wash in alcohol con- 

 taining powdered lithium carbonate. Roux's Method 

 for the destruction of cleavage spheres : Fertilize the 

 eggs of a frog (see Artificial Fertilization) and ro 

 minutes after the first cleavage begins, heat a needle 

 with a guard and intrpduce the point into the eggs, 

 above the equator and parallel to the cleavage. A 

 half-hour after the operation cover the dish, and after 

 another half-hour pour water over the eggs. They 

 may be examined in a few hours and on the next day. 

 Fix at successive stages of cleavage in chromic acetic 

 acid. Ruge's Stain for blood. Preparation of the 

 solution of polychrome methylene-blue. To a 1% 

 aojueous solution of methylene-blue add o. I ', Nat )]l. 

 Heat this solution — short of boiling — 3 or 4 times. 

 Add 0.2% NaOU. and repeat the heating. To make 



