• A droplet of deionised water is poured on a microscope slide. The bacterial cells are transferred with a bacteriological ring and an emulsion is formed. Next the droplet is spread at the centre of the slide by the ring.
• Cells are fixed by passing the slide over the flame of the Bunsen burner, without getting it overheated. During fixation process the slide should be sufferable when placed on the outside of the hand. The fixation is completed when the sample is completely dry.
• Afterwards, a few drops of crystal violet are added and the dye is left to take effect for 1 min. The crystal violet colourizes all the bacterial cells (without exception) with a deep blue colour.
• Then, the excess of dye is removed with water and sample is treated with Lugol's iodine (Ι2/ΚΙ) for 3 min. Next, the sample is treated with alcohol (a few droplets of it for 1-3 min.) and afterwards, with plenty of water. At this stage only gram-positive cells retain the blue colour, the rest of them are decolorized.
• The sample is covered with safranin (for 1-2 min) and then the excess of dye is washed off with water.
• The surplus of water is removed with absorbing paper, without dragging it on the surface of the slide. Next follows the microscopic observation at 40x magnification and afterwards, at 100x magnification (with oil submergence).
