ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 13 
SECTION 5: DIFFERENTIAL STAINING TECHNIQUE 
EXERCISE 18 
GRAM STAIN 
Make Gram stains of the cultures furnished. 
Procedure 
(1) Fix the preparation. 
(2) Stain with gentian violet for about 30 seconds. 
(3) Rinse with water. 
(4) Cover the film with Gram’s iodine and allow it to act for about 30 
seconds. 
(5) Rinse with water. 
(6) Decolorize in 95% alcohol. Thirty to 60 seconds is long enough for a 
thin smear. Decolorization should be continued until the color ceases to run from 
the film. 
(7) Rinse with water. 
(8) Counterstain with safranin or Bismark brown for ten to 30 seconds. 
(9) Rinse with water and blot dry. 
(10) Examine under the oil immersion objective. Make drawings. 
Gram positive organisms stain a purplish black; Gram negative organisms 
take the color of the counterstain. ; 
Formulae 
Gentian Violet Solution 
Gentian violet, saturated alcoholic solution. 10 ce, 
Ammonium oxalate, 1.0% aqueous solution 40 c.c. 
Gram’s Iodine Solution 
Iodine crystals 1 gram 
Potassium iodide 2 grams 
Distilled water. .....-300 cc. 
Bismark Brown Solution 
Saturated alcoholic solution “L1ee. 
Distilled water. 10 c.c. 
Safranin Solution 
Saturated aqueous solution 
