Hucker’s Modification Method Gram Staining Procedure

Principle:

The crystal violet stain is the primary stain, which stains everything in the smear blue. The Gram's iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranin is the counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus, gram-negative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells, and mucus will stain even lighter pink than the pus cells.

Procedure:

• Flood the smeared areas with CRYSTAL VOILET stain for 1 minute 
• Washe with water, and applies enough GRAMS IODINE for 1 minute 
• Rinse off with water and decolorizes with ACETONE-ALCOHOL solution until no further color flows from the slide. 
• Apply enough SAFRANIN stain for 1 minute then washes off with water 
• Air dry or blot between sheets of blotting paper and examine under oil immersion lens.

Gram Stain Interpretation:

Gram-positive organisms stain a deep blue,blue or purple.Gram-negative organisms stain a deep pink or pink, but since the background material is also pink, minute and detailed inspection is necessary before reporting the results.