Before you start

Use young cultures (18–24 h); older Gram-positive cells can stain Gram-negative.
Include known Gram-positive and Gram-negative control organisms.

Reagents and materials

Totals are for 4 slides (+10% extra). Per-slide amounts are in the middle column.

Reagent	Per slide	Total
Crystal violet	1 mL	1 mL
Gram’s iodine	1 mL	1 mL
Decolouriser (95% ethanol or acetone–alcohol)	2 mL	2 mL
Safranin	1 mL	1 mL
Immersion oil	1 drop	1 drop

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Prepare the smear
Spread a small amount of culture in a drop of water on a slide and let it air-dry.
Heat-fix
Pass the slide quickly through a flame 2–3 times (or use a slide warmer).
Crystal violet
Flood with crystal violet for 1 min, then rinse gently with water.
Iodine
Flood with Gram’s iodine for 1 min, then rinse.
Decolourise
Add decolouriser drop by drop for 5–15 s until the run-off is clear; rinse immediately.
Counterstain
Flood with safranin for 30–60 s, rinse and blot dry.
Examine
Examine under oil immersion: purple = Gram-positive, pink/red = Gram-negative. Note shape and arrangement.

Decolourisation is the critical step: too long makes Gram-positive cells look negative; too short leaves Gram-negative cells purple.

Problem	What to check or change
Everything purple	Decolourise longer; make thinner smears.
Everything pink	Shorter decolourisation; use fresh cultures.

Work at the organism’s biosafety level; stains are irritants.