Gram staining: Principle, Requirements, Procedure and Microscopic Examination

  • Gram staining is a differential staining technique that is used for microscopic examination of bacteria.
  • In differential staining, specimen is subjected to a series of stains (dyes) in which different organisms or different parts of the cell are stained differently so that they can be distinguished from each other.
  • Gram staining differentiates bacteria into two groups; Gram positive and Gram negative
  • It was developed by Hans Christian Gram in 1884 and modified by Hucker in 1921.


  • The difference in the chemical composition of bacterial cell walls accounts for the Gram staining differences between the two major groups of bacteria (Gram positive and Gram negative).
  • Gram-positive cell walls contain thick peptidoglycan layer with numerous teichoic acid cross linkages whereas, the peptidoglycan layer in Gram-negative cells is much thinner and surrounded by outer lipid containing layers.
  • Peptidoglycan, a polysaccharide composed of two chemical subunits, N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) are cross-linked by short chains of peptides by means of a trans-peptidase enzyme, resulting in the shape and rigidity of the cell wall.
  • In case of Gram-negative bacteria, the cross-linking of the peptidoglycan layer is direct because the bacteria do not have short peptide tails.
  • Gram-positive bacteria retain the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). Mordant is a substance that increases the cells’ affinity for a stain.
  • The iodine and crystal violet form a complex (CV-I) within the peptidoglycan. When a decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue.
  • In Gram-negative cells, following the application of the crystal violet and iodine, the CV-I complexes are not trapped within the peptidoglycan.
  • Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, and also dissolves the lipids leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells.
  • The cells appear colorless. Therefore, a counter stain, safranin, is applied, to make the cells distinctly visible (either red or pink).
  • Gram-positive organisms that have lost cell wall integrity because of anti-biotic treatment, old age, or action of autolytic enzymes may allow the crystal violet to wash out with the decolorizer and appear Gram-variable, with some cells staining pink and  others staining purple.


  1. Primary stain:2 gm Crystal violet, 20 ml 95% ethyl alcohol, 0.8 gm ammonium oxalate, and 100 mL distilled water
  2. Gram’s iodine (Mordant):2 gm potassium iodide, 1 gm iodine crystals, and 100 ml distilled water
  3. Decolorizer:Acetone and ethanol (50 ml each)
  4. Counterstain:0 gm Safranin, 200 ml 95% ethanol, and 800 ml distilled water
  5. Fresh culture sample: 24-hour agar culture of Staphylococcus epidermidis, 24-hour agar culture of Bacillus subtilis and 24-hour agar culture of Escherichia coli
  6. Bunsen burner
  7. Inoculating loop
  8. Microscope
  9. Distilled water or tap water
  10. Soft cotton or blotting paper
  11. Grease free glass slides


  1. Fix the material (any of the specimen or culture sample) on the clean grease free slide with methanol or heat. If slide is heat fixed, allow it to cool to the touch before applying stain.
  2. Flood the slide with crystal violet (purple) and allow it to remain on the slide without drying for 10-30 seconds.
  3. Rinse the slide with distilled water or tap water, shaking off all excess.
  4. Flood the slide with iodine (mordant) to increase the affinity of crystal violet and allow it to remain on the surface without drying for twice as long as crystal violet was in contact with the slide.
  5. Rinse the slide with tap water, shaking off all excess.
  6. Flood the slide with acid-alcohol decolorizer for 10 seconds and rinse immediately with tap water. Repeat this step until the blue dye no longer runs off the slide with the decolorizer. Thicker smears require more prolonged decolorizing . rinse with tap water and shake off excess.
  7. Flood the slide with counter stain, safranin and allow it to remain on the slide without drying for 30 seconds. Rinse with tap water and gently blot the slide dry with soft cotton or blotting paper or air dry. For delicate smears, such as certain body fluids, air drying is the best method.
  8. Examine microscopically under an oil immersion lens at 1000X.

Microscopic Examination:

  • Gram-positive cells :Blue/Purple Color e.g. Staphylococcus epidermidis, Bacillus subtilis
  • Gram-Negative cells :Red/Pink Color e.g. Escherichia coli

Gram staining: Principle, Requirements, Procedure and Microscopic Examination