14 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
EXERCISE 19 
ACID-FAST STAIN 
Make stained preparations of the cultures furnished and also of tubercular 
sputum. 
Procedure 
(1) Fix the slide. ; 
(2) Stain the fixed smear for five minutes by placing the slide in a recep- 
tacle containing carbol-fuchsin heated by boiling water. 
(3) Rinse with water. 
(4) Decolorize for one to three minutes in acid alcohol. 
(5) Wash thoroughly with water. 
(6) Counterstain with methylene blue for thirty seconds to one minute. 
(7) Examine under the oil immersion objective and make drawings. 
The tubercle organisms will be stained red. Any non-acid-fast organisms 
present will be stained blue. 
The carbol-fuchsin and methylene blue solutions used for this purpose are 
the same as those employed in previous exercises for simple stains. The acid 
alcohol solution contains 3% hydrochloric acid in 95% alcohol. 
EXERCISE 20 
SPORE STAIN 
Make stained preparations of the culture furnished. 
Procedure 
Follow the same procedure as outlined in Exercise 19, except in step (4). 
In step (4) decolorize in 95% alcohol until most of the color is removed. 
When this method is carried out successfully, the spores will have a distinct 
red color and the rest of the organisms will be blue. 
_ EXERCISE 21 
SPORE STAIN: DORNER’S METHOD 
Make stained preparations of the cultures furnished. 
Procedure 
(1) In five drops of sterile water in a test tube make a very heavy suspen- 
sion of the organism. 
(2) Add an equal quantity of carbol-fuchsin. 
(3) Allow to stain in boiling water bath for ten minutes. 
(4) From these stained organisms make a nigrosine preparation. 
(5) Examine under the oil immersion objective and make drawings. 
By this method the spores will have a red color and the rest of the organ- 
isms are seen unstained in a dark background. 
