Gram Staining – Its Principle, Procedure, and Results

Gram staining is a differential bacterial staining method used to distinguish between Gram Positive and Gram Negative bacteria based on the makeup of their cell walls.

There are many different types of staining the most important one is gram staining. It is both the most used and significant staining method in bacteriology, particularly in medical bacteriology. It is frequently the initial test carried out on bacteria during the process of their identification and observation.


     Hans Christian Gram, a Danish bacteriologist, developed this method in 1884. He created this staining method to find the pneumonia-causing bacteria. The division of bacteria into Gram-Positive and Gram-Negative kinds later gained popularity.

Principle of staining?

     Some bacteria can retain the primary stain after being stained with the primary stain Crystal Violet and fixed with the mordant, while other bacteria are decolored by alcohol. Gram-positive bacteria have low lipid contents and a thick covering of peptidoglycan, a protein-sugar compound.

   The cell’s thick cell wall becomes dehydrated and shrinks as a result of decolorization, which seals the cell wall’s pores and keeps the stain from leaving the cell. Because it is attached to the gram-positive bacteria’s substantial layer of peptidoglycan and looks blue or purple, the Crystal Violet-Iodine combination cannot be removed by ethanol.

   In the case of gram-negative bacteria, the CV-Iodine complex is likewise taken up by the cell wall, but because of the thin layer of peptidoglycan and a thick outer layer made of lipids, the CV-Iodine complex is washed off. Decolorizer dissolves the lipids in the cell walls when alcohol is present, allowing the crystal violet-iodine combination to leak out of the cells. Then, when dyed with safranin once more, they take the stain and turn red.


  • Bacterial culture
  • Gram Staining Kit (Reagents)
  • glass slide
  • Inoculating  loop
  • Bunsen burner staining rack
  • bottle cleaning (or Tap water)
  • 100x objective lens for a microscope (compound microscope)

Gram stain reagents

Primary Stain (Crystal Violet):

     Crystal violet is used as the main stain in the gram stain. Because this fundamental dye is positively charged, it binds to the cell membranes of both gram-positive and gram-negative cells. After applying crystal violet, the extra stain is wiped off with water after 60 seconds.

Mordant (Gram’s iodine):

       Iodine and potassium iodide are dissolved in water and employed as a mordant in the Gram staining process. The CVI complex that results from its interaction with CV+ becomes stuck in the Gram-Positive cell wall’s dehydrated peptidoglycan layer.

 Decolorizing solution:

      It is either acetone or ethanol (95%) or a 1:1 volume mixture of acetone and ethanol. The decolorizing solution enhances the Gram-Negative cell wall’s permeability by dissolving the outer membrane’s lipid content. In contrast, the decolorizer in the cell wall of Gram-positive bacteria dehydrates the peptidoglycan layer and confines the CVI complex inside the cell.

 Counter Stain (Safranin):

           It is a red counterstain used in the Gram Staining procedure to stain decolorized Gram-Negative cells. It is a simple dye that interacts with the negatively charged cell wall and membrane elements.

Gram stain procedure

  • With a loopful of the sample, prepare the smear of suspension on the clean slide.
  • Heat fix the slide and let it air dry.
  • Add drops of crystal violet to the smear for 30 to 60 seconds, and then rinse it with water.
  • Flood the Gram’s iodine for 1 minute, then rinses with water.
  • Add 95% alcohol for 10 to 20 seconds, and rinse with water
  • Add safranin for one minute, and rinse with water.
  • Air dry, wipe dry, and examine under a light microscope.

The procedure of light microscope observation?

  • Use stage clips to secure the slide with the Gram-stained, air-dried smear to the light microscope’s stage.
  • To direct light toward the smear, move the stage.
  • Utilizing a coarse adjustment knob, align the 10X objective lens and concentrate on the smear.
  • Set the objective lens to 40X and use the fine adjustment knob to focus.
  • To get the smear to fall between a 40X and 100X objective, rotate the nose piece.
  • A drop of immersion oil should be added over the smear.
  • Align the 100X oil immersion objective lens over the smear by rotating the nosepiece.
  • Study the microorganisms under the microscope by using a little adjustment knob.

Gram stain results and interpretation

    Gram-Positive Bacteria change color to purple or blue in color.

    Gram-negative bacteria change color to pink or red.


Rimsha Bashir
Rimsha Bashir

Rimsha Saith is a highly knowledgeable microbiologist with a keen interest in the field. Her expertise and passion are in her writing for Microbiology. As a writer, Rimsha has authored numerous articles that have been well-received by both health and medical students and industries.

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