i8o 
PHARMACEUTICAL BOTANY 
handle permits the placing of the forceps on the table while the cul- 
ture material is obtained. 
3. Place several drops of distilled water on the cover slip and add a 
loop full of the organism secured from the pure culture in a test tube 
as follows: 
4. Remove the cotton plug by the third and fourth fingers of the 
left hand. 
5. Hold the open test tube between the thumb and forefinger of the 
left hand. 
6. By means of a previously flamed platinum needle, remove a 
little of the culture from the surface of the culture media. 
7. Replace the cotton plug. 
8. Add the culture media to the drop of distilled water on the 
cover slip and distribute this material by stirring. 
9. Evaporate the water on the cover slip to dryness by holding it 
some distance above the Bunsen flame and slowly enough to prevent 
connection circles being formed by the material affixed to the cover. 
10. Pass the cover glass three times through the Bunsen flame. 
11. Apply the stain, which should remain long enough to stain the 
objects. 
12. Wash off the stain with distilled water. 
13. Dry the cover glass above the flame. 
14. Apply a drop of balsam, turn the cover slip over and drop it on 
to the center of a glass slide previously provided and cleaned for this 
purpose. 
Gram’s Method. — This is a method of differential bleaching after 
a stain. The cover glass preparations or sections are passed from 
absolute alcohol into Ehrlich’s anilin gentian violet, where they 
remain one to three minutes, except tubercle bacilli preparations 
which remain commonly twelve to twenty-four hours. They are 
then placed for one to three minutes (occasionally five minutes) in 
iodine potassium iodide water (iodine crystals 1, potass, iodide 2, 
water 300), with or without washing lightly in alcohol. In this 
they remain one to three minutes. They are then placed in absolute 
alcohol until sufficiently bleached, after which they are cleared in 
clove oil and mounted in balsam. 
Certain organisms, when stained by this method give up the stain 
