Further Studies on a Modification of the Gram Stain

Abstract

In perfecting the modification of the Gram-stain previously proposed, the following points are of interest: 1. Acetone is too strong a decolorizer for Gram-positive organisms and alcohol too weak for Gram-negative organisms. Consequently, it is now recommended that equal parts of acetone (100% c.p.) and ethyl alcohol (95%) be used as a decolorizing agent. The time of application should not ordinarily exceed 10 seconds. 2. Aqueous basic fuchsin (0.1%) serves as a strongly contrasting counterstain. Prolonged application renders Gram-positive organisms doubtful or Gram-negative, while short application renders Gram-negative organisms doubtful or Gram-positive. Twenty (20) seconds is therefore recommended as the time of application of the counterstain. 3. The method here described, with due regard for its limitations, is of value in Gram-staining pure or mixed cultures as well as for organic materials, such as Acidophilus milk, feces, etc., either for research purposes or classroom use. The method is as follows: Air-dry film and fix with least amount of heat necessary. Flood with dye for 5 minutes. Previously mix 30 drops of a 1% aqueous solution of crystal violet or methyl violet 6B with 8 drops of a 5% solution of sodium bicarbonate. Allow the mixture to remain for 5 minutes or more. Flush with iodine solution for 2 minutes. Two grams iodine dissolved in 10 cc. normal sodium hydroxide solution and 90 cc. water added. Drain without blotting but do not allow film to dry. Add a mixture of equal parts of acetone and alcohol drop by drop until the drippings are colorless. (10 seconds or less.) Air-dry slide. Counterstain for 20 seconds with 0.1% aqueous solution of basic fuchsin. Wash off excess stain by short exposure to tap water and air-dry. If slide is not clear immersion in xylol is recommended.