152 PATHOGENIC BACTERIA. 
(1) For cultures use blood-serum, agar, and bouil- 
lon to which from 4 to 8 per cent. of glycerin has 
been added. 
(2) Stain films, prepared from cultures, with ordi- 
nary stains’ and by the Ziehl-Neelson method as 
follows : 
(a) Prepare films as usual. 
(4) Stain in Ziehl’s carbol-fuchsin, steaming but 
not boiling, five minutes ; cold, twenty minutes. 
(c) Wash in 20 per cent. sulphutic acid for three 
to five seconds. 
(2) Wash in 60 per cent. alcohol until no red 
color is left. 
(e) Wash in water. 
(f) Stain in aqueous solution of methylene-blue 
for one minute. 
(g) Wash in water, dry, and mount. 
The bacteria of leprosy, syphilis, and smegma 
stain by this method. Bacterium lepre stains 
much more quickly than B. tuberculosis, B. syphil- 
idis is decolorized more quickly, especially in sul- 
phuric acid, and B. smegmatis is decolorized by the 
alcohol. 
2. Examination of Tuberculous Sputum. 
(1) Ziehl-Neelson Method. 
(a) Place the sputum in a shallow glass dish on 
a black surface. 
(4) Select several of the characteristic yellowish 
1The tubercle bacillus takes the ordinary stains very 
slowly and faintly. 
