Gram stain

Principle

Gram-positive bacteria have a thick peptidoglycan wall that traps the crystal violet–iodine complex, so they resist decolourisation and stay purple. Gram-negative bacteria have a thin peptidoglycan layer and an outer lipid membrane; the decolouriser removes the dye and they take up the red counterstain.

Reagents

• Crystal violet (primary stain)
• Gram's iodine (mordant)
• Decolouriser: acetone-alcohol (or acetone alone; iodine-acetone in the Preston–Morrell method)
• Counterstain: safranin or dilute carbol fuchsin

Procedure

• Make a thin smear, air-dry and heat- or methanol-fix.
• Crystal violet 30–60 s, rinse with water.
• Iodine 30–60 s, rinse.
• Decolourise briefly (a few seconds) until colour stops running, then rinse at once.
• Counterstain 30–60 s, rinse, blot dry and examine under oil immersion.

Results

• Gram-positive: dark purple
• Gram-negative: pink to red
• Yeasts: purple; pus cell nuclei: red

Quality control and pitfalls

• Controls: Staphylococcus aureus (positive) and Escherichia coli (negative).
• Over-decolourising makes positives look negative; thick smears and old cultures (older than 24 h) can also give false Gram-negative results.

Report

Quantity of cells and bacteria (few, moderate, many), Gram reaction, shape and arrangement, and whether organisms are inside pus cells.