METHODS FOR MICROSCOPIC STUDY OF BACTERIA 207 
Solution I — Glacial acetic acid 1 cc. 
Formalin, 40 per cent (neutral) 20 cc. 
Distilled water 100 cc. 
Solution II — Tannic acid 5 gm. 
Distilled water 100 cc. 
Solution III — (Prepare fresh each time.) Add 10 per cent aqueous ammonia solu- 
tion to a 5 per cent AgNOa solution, drop by drop, until a brown 
precipitate forms and redissolves. Then add silver nitrate until a 
faint opalescence develops, persisting after shaking. 
Tn stain: 
(a) Spread smear very thin, and air-dry. 
(b) Wash with sokition I for one minute, replacing sohition at least 
twice. 
(c) Wash with water. 
(d) Add solution II, and steam thirty seconds. 
(e) Wash thoroughly with water. 
(/) Add solution III; steam gently for thirty seconds. 
(g) Wash thoroughly in water. 
Immersion oil slowly decolorizes the spirochetes which appear almost 
black, due to impregnation with silver. 
5. W. H. Smith's Sputum Stain. — (o) Stain the fixed smear with 
anilin oil gentian violet for one minute. 
(6) Wash with water. 
(c) Flood with Gram-iodine solution for thirty seconds. 
(d) Decolorize with 95 per cent alcohol. 
(e) Wash with ether for a few seconds. 
(/) Flood with absolute alcohol for five seconds. 
((/) Stain with saturated aqueous solution of eosin for one to two 
minutes. 
(h) Wash with absolute alcohol for a few seconds. 
(i) Clear with xylol. 
(•*) Mount in balsam. 
III. Staining Bacteria in Tissues. — Paraffin sections are preferable, 
partly because very thin sections may be cut; chiefly because celloidin 
stains somewhat with the stains ordinarily used. 
The (Iram-Weigert Stain for Bacteria in Tissves.^ — {a) Stain paraffin 
sections with anilin oil methyl violet for five to twenty minutes. 
(6) Wash in water to remove excess of stain. 
(c) Gram-iodine solution for one minute. 
(d) Wash in water to remove excess of iodine. 
(e) Decolorize with several changes of absolute alcohol until no 
more color comes out. 
(/) Clear section in xylol. 
(g) Mount in neutral xylol balsam. 
Mallory and Wright Modification for Celloidin Sections.-— (a) Stain 
sections with lithium carmine for two to five minutes. 
1 Mallory and Wright: Pathological Technic, 6th ed., 1915, p. 432. " Ibid. 
