The Processes Involved In Gram Staining

What Exactly is Gram Staining?

Gram staining is a common technique for distinguishing two large groups of bacteria based on differences in cell wall constituents.

Gram staining differentiates between Gram positive and Gram negative groups by coloring cells pink or violet.

Gram-positive bacteria stain violet because their cell walls contain a thick layer of peptidoglycan, which retains the crystal violet with which they are stained.

Gram negative bacteria, on the other hand, stain pink due to a thinner peptidoglycan wall that does not retain the crystal violet during the decoloring process.

What Is the Process of Gram Staining?

Gram staining consists of three steps:

  1. Staining with crystal violet, which happens to be a water-soluble dye.
  2. Decolorization, and
  3. Counterstaining, usually done with safranin.

Gram positive bacteria (with a thicker peptidoglycan layer in the cell membrane) retain crystal violet stain during the decolorization process, whereas Gram negative bacteria lose the crystal violet stain and are stained by safranin in the final staining process due to differences in the thickness of the peptidoglycan layer in the cell membrane.

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The procedure consists of three steps:

  1. Crystal violet dye is used to stain the cells.
  2. The crystal violet and iodine complex is then formed by adding a Gram’s iodine solution (iodine and potassium iodide). This complex is insoluble in water and has a larger molecule than the original crystal violet stain and iodine.
  3. The sample is decolorized with ethyl alcohol or acetone, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex cannot penetrate the tightened peptidoglycan layer and is thus trapped in Gram positive bacteria cells.
  4. In contrast, Gram negative bacteria’s outer membrane is degraded, and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex, resulting in color loss.
  5. The sample is stained red by the addition of a counterstain, such as the weakly water soluble safranin. Safranin does not interfere with the purple coloration of Gram positive cells because it is lighter than crystal violet. The decolorized Gram negative cells, on the other hand, are stained red.
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How To- Staining Protocol:


  1. Violet crystal (primary stain)
  2. Gram’s Iodine/Iodine solution (mordant that fixes crystal violet to cell wall)
  3. Decolorizer (e.g. ethanol)
  4. Safranin (secondary stain)
  5. Water (preferably in a squirt bottle)


  1. Make a slide of the stained cell sample. Heat fix the sample to the slide by carefully passing it through a Bunsen burner three times with a drop or small piece of sample on it.
  2. Incubate the sample/slide for 1 minute with the primary stain (crystal violet). To remove unbound crystal violet, rinse the slide with a gentle stream of water for no more than 5 seconds.
  3. Add Gram’s iodine for one minute—this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall.
  4. Rinse the sample/slide for 3 seconds with acetone or alcohol, then rinse with a gentle stream of water. If the sample is Gram negative, the alcohol will decolorize it, removing the crystal violet. However, if the alcohol is left on the sample for an extended period of time, it may decolorize Gram positive cells.
  5. Incubate the slide for 1 minute with the secondary stain, safranin. Wash for a maximum of 5 seconds with a gentle stream of water. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) while rejecting the secondary stain (safranin), resulting in a violet/purple appearance under a microscope. If the bacteria is Gram negative, it will absorb the secondary stain instead of the primary stain, causing it to appear red under a microscope.
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Frequently Asked Questions

What is the most crucial aspect of a Gram stain?

The decolorizing step is the most important in determining the outcome of the stain.

Which reagent is the most important in the Gram stain method?

Crystal violet is the primary stain used in Gram’s method. Methylene blue, which is equally effective as crystal violet, is sometimes used in its place.

What is the underlying principle of Gram staining?

Gram staining is based on the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.