METHODS FOR MICROSCOPIC STUDY OF BACTERIA 205 
6. Pour off alcohol, flood with neutral red solution. Allow to stain 
one minute. 
7. Wash in water, dry and examine. 
Do not use water until Step 7 is reached. 
2. Stains for Acid-fast Bacteria.— Ziehl-Neelsen Method.^— (a) Stain 
dried and fixed smear with carbol fuchsin, as described on page 199. 
(b) Wash thoroughly with water. 
(c) Decolorize with acid alcohoP until no more color can be 
washed out. 
(d) Wash with water. 
(e) Counterstain lightly with Loffler's alkaline methylene blue. 
(/) Wash, dry and mount. 
Acid-fast bacilli and spores stain red; all others blue. 
3. Frdnhel-(kihhet Met}wd.^—{a) Stain with carbol fuchsin as in 
the Ziehl-Xeelsen method and wash in water. 
{h) Decolorize and counterstain simultaneously with the following 
solution: 
Methylene blue 2 gm. 
Water 75 cc. 
Sulphuric acid 25 cc. 
The counterstain is allowed to act for one minute. 
(c) Wash, dry and mount. 
4. Polychrome *S7ai/?5.— Polychrome stains are of special value for 
the examination of exudates, body fluids or tissues in w^hich the his- 
tological relations of bacteria are to be investigated. These stains, 
or modifications of them, are also useful in the study of Treponemata, 
Spirochetes and Protozoa. 
Wright's Stain.'^—Preixtratio?}. — "To a 0.5 per cent aqueous solu- 
tion of sodium bicarbonate add methylene blue (B.X., or 'medi- 
cinally pure') in the proportion of 1 gm. of the dye to each 100 cc. of 
the solution. Heat the mixture in a steam sterilizer at 100° C. for 
one full hour, counting the time after the sterilizer has become thor- 
oughly heated. The mixture is to be contained in a flask, or flasks, 
of such size and shape that it forms a layer not more than 6 cm. deep. 
After heating, the mixture is allowed to cool, placing the flask in cold 
water if desired, and is then filtered to remove the precipitate which 
has formed in it. It should when cold have a deep purple-red color 
when viewed in a thin layer by transmitted yellowish artificial light. 
It does not show this color while it is warm. 
"To each 100 cc. of the filtered mixture add 500 cc. of a 0.1 per 
cent aqueous solution of 'yellowish, water-soluble' eosin and mix 
thoroughly. Collect on a filter the abundant precipitate which 
1 Ziehl: Deutsch. med. Wchnschr., 1882, 8, 451; 1883, 9, 62, 247. Neelsen: Cen- 
tralbl. f. med. Wissensch., 1883, p. 600. 
2 Ninety per cent alcohol containing 3 per cent by volume of hydrochloric acid. 
3 Frankel: Berlin, khn. Wchnschr., 1884,21. 193, 214. Gabbet: Lancet, 1887, i, 757. 
^ Mallory and Wright: Pathological Technic, 1915, 6th ed., p. 382. 
