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How Gram Stain works? Gram Staining Principle: Step by step procedure with explanation

Gram Staining Principle:
Gram Staining Procedure

Step 1: Prepare thin smear of bacteria on a clean slide

Step 2: Cover the smear with few drops of crystal violet for 1 min

Step 3: Wash excess stain with water

Step 4: Add few drops of iodine solution on the smear for 1 min

Explanation: Here Iodine solution is a mordant. Mordants are substance that improves the staining property of the dye. Here Iodine favors more interaction between cell and the dye (crystal violet). So that, the cells are more strongly stained.

Step 5: Rinse in water
Explanation: now under the microscope, all cells are violet coloured we cannot differentiate Gram positive and Gram negative bacteria.

Step 6: Wash with 95% ethanol for a minute.
Explanation: On washing with ethanol or acetone, the smear either retains the violet colour or decolorizes. Those bacteria that retain violet colour are Gram positive, and those that lose violet colour on alcohol treatment are the Gram negative.
Gram Staining procedure

This difference in staining is due to the difference in the cell wall components of Gram positive and Gram negative bacteria.
How Gram stain works?
To answer this question, we have to have a close look at the cell wall layers of these bacteria. Look at the figure and see the difference in the cell wall layers of Gram positive and Gram negative bacteria.
Gram positive and Gram Negative  cell wall

  •           In Gram positive bacteria cell wall is made up of 2 layers.
  •          A thicker outer layer made of peptidoglycan and the cell membrane
In Gram negative bacteria, cell wall has three layers
  •       A lipid rich outer wall
  •      A middle layer consists of thin layer of peptidoglycan and cell membrane.
What happens to Gram negative bacteria on ethanol treatment?
Before ethanol treatment, the purple colour in Gram negative bacteria is mainly due to the staining of outer lipid layer and thin peptidoglycan wall with crystal violet.
On ethanol treatment, lipids are dissolved in ethanol and peptidoglycan wall is very thin to hold the stain so gram negative bacteria lose violet colour.

What happens to Gram positive bacteria on ethanol treatment?
In Gram positive bacteria, the lipid content is very low and has a thick peptidoglycan wall. So ethanol cannot remove the crystal violet stain bound to thick peptidoglycan wall of gram positive bacteria.

Step 7: Rinse with water

Step 8:  Stain with 1% safranin for 1 minute.
Explanation: This is to visualise Gram negative bacteria. After ethanol treatment in step-6, only Gram positive bacteria retain crystal violet. We need to stain further to visualise Gram negative bacteria.

Step 9: Wash stain in excess water.
The end result after staining with safranin :
Gram Positive
Gram Positive Bacteria
Gram Negative
Gram Negative Bacteria

  •  Gram positive cells appear violet or purple (Colour of crystal violet)
  •    Gram negative cells appear pink to red (colour of safranin).

Reagent Preparation
  • Crystal violet solution: 1g crystal violet dissolved in 100 ml ethanol
  • Safranin solution: 1g safranin dissolved in 100 ml H2O.
  • Iodine solution: Dissolve 2g potassium iodide (KI) in 100 ml water. To the solution add iodine crystals till a deep yellow colour develops.
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