GENERAL BACTERIOLOGY 
89 
B. Stain for Spores.— 
1. Make and fix the preparation in usual manner. 
2. Cover with Loeffler’s alkaline methylene blue and 
heat the stain until it boils, remove it from flame, then 
heat again; repeat this for one minute (Loeffler’s alkaline 
methylene blue is prepared by mixing 30 c.c. of saturated 
alcoholic solution of methylene blue with 100 c.c. of 
1:10,000 solution of potassium hydroxide in water). 
3. Rinse in water. 
4. Decolorize with a mixture of 98 c.c. of 80 per cent 
alcohol and 2 c.c. of nitric acid, until all blue has dis¬ 
appeared. 
5. Rinse in water. 
6. Dip three to five seconds in a mixture of 10 c. c. 
of saturated alcoholic solution of eosin and 90 c.c. of 
water. 
7. Rinse in water and blot. 
Spores are stained blue and the body of bacteria are 
stained pink. (See Fig. 4.) 
C. Stain for Capsule.— 
1. Smear the bacteria in a drop of beef blood serum, 
dry and fix by heat in the usual manner. 
2. Stain for a few seconds with a mixture of 5 c.c. of 
saturated solution of gentian violet and 95 c.c. of dis¬ 
tilled water. Hold the preparation flooded in this over 
a flame until it steams. 
4. Wash off the stain with 20 per cent aqueous solu¬ 
tion of copper sulphate. 
5. Blot.(do not wash in water). 
The capsule appears as a blue halo around a dark pur¬ 
ple cell body. (See Fig. 2.) 
D. Stain for Flagella.— (See Fig. 3.) 
Use bacterial emulsion from young cultures grown on 
agar media (see section on Culture Media). 
