90 
PRINCIPLES OF BACTERIOLOGY 
1. Dry the preparation in air and fix by heat. 
2. Pour the following mixture, freshly filtered: 10 c.c. 
of 20 per cent aqueous solution of tannic acid, 5 c. c. of 
saturated aqueous solution of ferrous sulphate, and 1 c. c. 
of saturated alcohol solution of fuchsin. 
Allow this mixture to remain thirty to forty-five sec¬ 
onds, heating it gently. 
3. Wash thoroughly in water. 
4. Stain for one to two minutes, warming gently, with 
the following freshly filtered mixture: 
50 c.c. of 5 per cent anilin gentian violet. 
0.05 gram of sodium hydroxide. 
5. Wash in water and blot. 
E. Stain for Tuberculosis and Other “Acid-Fast” 
The Ziehl-Neelsen Method. 
1. Make the preparation (from the culture, urine, 
sputum, etc.) in the usual manner, dry in air and fix with 
heat. 
2. Pour the carbol-fuchsin stain (its preparation has 
been given in the section on Gram’s Method), and heat 
gently until it steams; continue this for three to five 
minutes, this had best be done on a special staining 
support under which there is a gas pipe with numerous 
perforations, connected by a piece of rubber tubing with 
the source of gas supply; this method permits of regu¬ 
lating the amount of heat just sufficient for steaming, 
thus preventing the boiling. (See Fig. 15.) 
3. Decolorize with 1 per cent hydrochloric acid or 5 
per cent nitric acid in alcohol, then with 90 per cent 
alcohol until no pink color is discharged. 
4. Wash in water. 
5. Stain in aqueous saturated solution of methylene 
blue for one to two minutes. 
6. Rinse in water, and blot. 
