STAINS 



502 



STAINS 



harden in alcohol. Stain with Heidenhain's iron- 

 hematoxylin. 4. For spores and tubercle bacilli : 

 Stain the cover-glass preparation with carbol-fuchsin, 

 wash in 60^ alcohol, then in water. Treat for 15 

 minutes with a 5% or lof c solution of potassium car- 

 bonate (or for a shorter time with the hydrogen dioxid) 

 and counterstain with methylene-blue. Murbach's 

 Method for preserving the transparency of the lens : 

 Open the eyeball, detach the lens and expose it to the 

 vapor of formalin. It will become hard and dry with- 

 out losing its clearness. Nakanishi's Method for 

 staining bacteria and the plasmodium malariae: Dis- 

 solve methylene-blue to saturation in hot water. 

 Spread a few drops on a slide, by means of a piece of 

 filter-paper, and then wash off the dye, so that only 

 enough remains to give the glass a sky-blue color. 

 Put a drop of blood or of a bacterial suspension on the 

 slide and apply a cover-glass. This method of stain- 

 ing is said to demonstrate the finer structural details of 

 the organism. Neelsen-Johne Method for staining 

 tubercle bacilli : Dry the films at room- temperature 

 or for 5 minutes in the oven at 75° C. and spray them 

 for 2 or 3 minutes with equal parts of alcohol and 

 ether; then stain with steaming hot carbol-fuchsin and 

 decolor for 2 minutes with 2.5 c / sulfuric acid or for one 

 minute with 20% picro-sulfuric acid ; wash in water 

 and stain one minute in dilute aqueous solution of 

 malachite green. Place the slide in the oven at 6o° C. 

 for a few minutes and while still warm put a few drops 

 of cedar oil on the film. When cold, examine without 

 a cover-glass. Neisser's Method. 1. For micro- 

 tome sections of gelatin or agar cultures of bacteria : 

 Fix for from I to 8 days, according to the size of the 

 object, in I °/c potassium bichromate, exposing mean- 

 while to the light. Wash in water and harden in alco- 

 hol. Stain with any of the usual anilin dyes. Alco- 

 hol or anilin will extract the color from the gelatin and 

 the agar. 2. For the pole granules of the bacilli of 

 diphtheria : Stain for 3 seconds in a mixture of 

 methylene-blue I gm., 90% alcohol 20 c.c, glacial 

 acetic acid 50 c.c, distilled water 350 c.c; wash in 

 water and stain for 5 seconds in filtered aqueous solu- 

 tion of bismarck brown 2 : icod, prepared by boiling. 

 Wash, dry, and mount in balsam. Neusser-Ehrlich 

 Stain: Prepare concentrated aqueous solutions of 

 methyl green, orange G, and acid fuchsin (extra) and 

 daily add more of the dye until an undissolved residue 

 remains. Then mix 50 c.c. of the acid fuchsin, 70 c.c. 

 of the orange G, and 8o c.c. of the methyl green and 

 add 150 c.c of distilled water, 80 c.c. of absolute 

 alcohol, and 20 c.c. of glycerol. Keep in the dark 3 

 weeks before using. Neutral Red, a metachromatic 

 basic dye. The term "neutral" refers to the tint of 

 its solution. It is used for intra vilam staining, in the 

 same way as methylene-blue, and in I °/o or stronger 

 aqueous solution for sections of fixed tissues. Its neu- 

 tral hue is turned bright red by acids, yellow by 

 alkalis. It stains mucin and cytoplasmic granules. 

 Nicholl-Rieder Method. See A'ieder's Method. 

 Nicolas' Method for hygroscopic material : Soak 

 the object for 2 days in a 4% aqueous solution of gela- 

 tin at 25 C.J for 2 days in a IO% solution; then for 

 2 days in a 25% solution containing 10% of glycerin 

 and kept at 35 C. Embed in the same mass in a 

 paper tray and when the gelatin sets harden in 5% 

 formalin. Preserve in weak formalin, dilute alcohol 

 or glycerin, or water. The curling of the sections in 

 alcohol is corrected by cresylol. Nicolle's Method. 

 I. For bacteria: Stain for a minute in a mixture of 10 

 volumes of a saturated solution of thionin in 50% 

 alcohol, and loo volumes of I '/, carbolic acid. 2. 

 For the capsules of Friedlander's bacillus: Stain for 



a few seconds in carbol-gentian-violet and immediately 

 transfer to a mixture of alcohol, 2 parts, and acetone, 

 I part. 3. For bacteria that have been decolored 

 by the method of Gram. Counterstain in Loffler's 

 methylene-blue, decolor in acidulated water, wash, 

 and treat for an instant with 10% solution of tannin. 

 Five c.c. of a saturated alcoholic solution of fuch- 

 sin diluted with 100 c.c. of water may be used. 

 4. See Grant's Method. Niessing's Fluid for 

 fixing mitotic figures: I. Platinum chlorid, 10% 

 solution, 25 parts, 2% osmic acid 20 parts, gla- 

 cial acetic acid 5 parts, distilled water 50 parts. 2. 

 The same with saturated aqueous solution of mercuric 

 chlorid instead of water. Night Blue, so named 

 because it shows as well in artificial light as in sun- 

 light. The following formula is recommended for 

 staining flagella : Ten c.c. of concentrated alcoholic 

 solution of night blue, 10 c.c. of 10% solution of 

 alum, and 10 c.c. of 10% solution of tannin. 

 Nigranilin, the same as the anilin black of Lightfoot. 

 Nikiforoff's Method for spirilla of recurrent fever: 

 Fix for 24 hours in a mixture of equal parts of 5% 

 potassium bichromate solution and saturated solution 

 of mercuric chlorid mo.6fc sodium chlorid solution; 

 harden in the oven in 70%, 80^, and 95% alcohols; 

 embed in paraffin ; stain for 24 hours in a mixture of 1 c / c 

 alcoholic tropseolin solution 5 c.c, saturated aqueous 

 solution of methylene-blue 10 c.c, caustic potash' 

 (1 : looo) 2 drops; wash in water; dip 2 or 3 times in 

 a mixture of equal parts of alcohol and ether; berga- 

 mot oil, xylol, balsam. Nissl's Method for gan- 

 glion-cells: I. Stain sections of tissue hardened in 

 \O c /c formalin or in graded alcohols in hot concentrated 

 aqueous fuchsin solution. 2. Stain in hot 0.5^ 

 methylene-blue ; when cool transfer to a mixture of 

 anilin (20 parts) and 90% alcohol (200 parts); then 

 treat with origanum oil, then with benzine, and mount 

 in solution of colophonium in benzine. 3. Mount 

 the hardened tissue on cork (without embedding), cut 

 sections and stain them in hot Nissl's methylene-blue 

 (see Staining Reagents) ; treat with the anilin alcohol 

 mixture, then with cajeput oil, then as in 2. Nissl's 

 Methylene-blue: Methylene-blue (B patent) 3.75 

 parts, Venice soap 1. 75 parts, distilled water looo 

 parts. Nitrosoindol Reaction: Add to a bouillon 

 culture of cholera bacilli of 24 hours a few drops of 

 pure concentrated sulfuric acid. The reaction is indi- 

 cated by a rose or purple-red color, of progressive in- 

 tensity, the older the culture. Nocht's Method for 

 staining the malarial parasite, and structural chromatin 

 in other microorganisms: Fix the film by heat or in 

 alcohol and stain for from 2 to 24 hours in Nocht's 

 stain (a. v.). Result: cytoplasm blue, chromatin 

 deep red, erythrocytes light pink. Nocht's Stain 

 for blood: Original method: Unna's polychrome 

 methylene-blue is neutralized with dilute acetic acid. 

 Solution A.— I c.c. of this neutralized polychrome 

 methylene-blue is mixed in a watch crystal with a sat- 

 urated aqueous solution of ordinary methylene-blue 

 until its red color disappears, and the solution becomes 

 blue. Solution B. — Dilute 3 drops to 4 drops of 1% 

 aqueous solution of eosin with 1 c.c. or 2 c.c. water. 

 Add solution A drop by drop to solution 1! until B is 

 dark blue ; a precipitate has then been formed. In 

 this mixture blood-films are lo be stained for several 

 hours up to 24 hours. Fix films in alcohol or by heat. 

 Subsequent modification: Solution A. — The poly- 

 chrome methylene-blue solution. To a 1 '',- aqueous 

 solution of methylene-blue add \.of c oro.5^rNa 2 C0 3 . 

 Heat at 50 C. to 6o° C. for several days. Solution 

 B. — Dilute 2 or 3 drops of 1 % aqueous solution of 

 eosin with I c.c. or 2 c.c. water. To solution B add 



