Required reagents: . Gram’s Iodine . Acetone-Alcohol . Safranin
Prepare the smear from the culture or from the specimen.
Allow the smear to air-dry completely.
Rapidly pass the slide (smear upper most) three times through the flame.
Cover the fixed smear with crystal violet for 1 minute and wash with distilled water.
Tip off the water and cover the smear with gram’s iodine for 1 minute.
Wash off the iodine with clean water.
Decolorize rapidly with acetone-alcohol for 30 seconds.
Wash off the acetone-alcohol with clean water.
Cover the smear with safranin for 1 minute.
Wash off the stain wipe the back of the slide. Let the smear to air-dry.
Examine the smear with oil immersion objective to look for bacteria.
Interpretation: . Gram-positive bacterium ……………Purple . Gram-negative bacterium …………..Pink