Gram Stain- Principle, Reagents, Procedure, Steps, Results

The Gram stain was developed by Christian Gram in 1884 and modified by Hucker in 1921.

The objective of Gram Stain

This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and (2) Gram-negative microorganisms that take the color of the counterstain (usually Safranin O).

Principle of Gram Stain

The two major groups of bacteria can be divided into gram-positive and gram-negative. The Gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups.

Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a complex within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue.

The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes. 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, leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink.

Gram Staining Procedure
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Gram Stain Reagents

Primary stain: 2 g Crystal violet, 20 mL 95% ethyl alcohol, 0.8 g ammonium oxalate, and 100 mL distilled water.
Gram’s iodine: 2 g potassium iodide, 1 g iodine crystals, and 100 mL distilled water.
Decolorizer: 50 mL acetone and 50 mL ethanol.
Counterstain: 4.0 g Safranin, 200 mL 95% ethanol, and 800 mL distilled water

The procedure/steps of Gram Stain

  1. Prepare and fix the specimen to the microscope slide before staining.
  2. Cover the smear with crystal violet, the primary stain, for 20 seconds.
  3. Gently rinse off the stain with water.
  4. Cover the smear with Gram’s iodine, the mordant, for 1 minute.
  5. Pour off the excess Gram’s iodine.
  6. Run the acid-alcohol decolorizer over the smear until the solution appears clear.
  7. Gently rinse with water.
  8. Cover the smear with safranin, the secondary or counterstain, for 20 seconds.
  9. Gently rinse the stain with water.
  10. Blot dry with bibulous paper.

Result Interpretation of Gram Stain

Gram Stain- Principle, Reagents, Procedure and Result Interpretation

Gram-positive: Blue/Purple Color
Gram-Negative: Red/Pink Color


  1. Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results.
  2. Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult. Gram-negative organisms may not decolorize properly.
  3. Cultures older than 16 to 18 hours will contain living and dead cells. Cells that are dead will be deteriorating and will not retain the stain properly.
  4. The stain may form a precipitate with aging. Filtering through gauze will remove excess crystals.
  5. Gram stains from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity due to the successful treatment.
  6. Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will not grow in culture. Some strains are obligate anaerobes.
  7. Toxin-producing organisms such as Clostridia, staphylococci, and streptococci may destroy white blood cells within a purulent specimen.
  8. Faintly staining Gram-negative organisms, such as Campylobacter and Brucella, may be visualized using an alternative counterstain (e.g., basic fuchsin).


Gram-Positive: Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Clostridium, etc.
Gram-Negative: E. coli, Salmonella Typhi, Shigella spp, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, etc.

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Gram Stain- Principle, Reagents, Procedure, Steps, Results

8 thoughts on “Gram Stain- Principle, Reagents, Procedure, Steps, Results”

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