Staphylococcus aureus Gram Stain: Introduction, Principle, Procedure and Result Interpretation

Staphylococcus aureus in single, pairs and clusters


Staphylococcus aureus in single, pairs and clusters in Gram stain as shown above picture. Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

Principle of  Gram stain

Principle of Gram stain on the various basis, like permeability of cell wall of bacteria and formation of dye-iodine complex, affinity, pH of the cytoplasm of organisms and presence of magnesium ribonucleate  as follows:

Permeability of cell wall of bacteria and formation of the dye-iodine complex (lake): The permeability of the cell wall of Gram-negative bacteria is more porous compared to the Gram-positive bacterial cell wall. The formation of the lake takes place in the cytoplasm of bacteria. The decolorizer ( alcohol) can not pass through the cell wall of Gram-positive bacteria and hence these cells become purple or violet whereas the Gram-negative bacterial cell wall permits the above decolorizer into the cell. Since the complex is soluble in organic solvent and insoluble in water, the dye-iodine complex will be washed away from the cell. As a result, these cells, now, are colorless and will take the color of counterstain, i.e. safranin or neutral red or dilute carbol fuchsin and appear pink/ red as shown in negative control as shown above picture.


Acidic substances have an affinity to basic dye and basic substances have an affinity to acidic dye.

The pH of the Cytoplasm of 0organisms

pH could be another factor that researchers have shown to describe the principle of Gram stain. The pH of the cytoplasm of Gram-positive bacteria is 2-3 whereas that of Gram-negative bacteria is 4-5. The acidic nature of the cytoplasm is further increased after the addition of iodine. Thus, it is the acidity of the cytoplasm of Gram-positive bacteria, which has an affinity to basic dye.

Presence of Magnesium ribonucleate 

Magnesium ribonucleate in the Gram-positive bacteria has an affinity to basic due. As a result, Gram-positive bacteria take the color of crystal violet. It has also been proved that these bacteria become Gram-negative after removal of Magnesium ribonucleate.

Requirements for Staining Staphylococcus  aureus

a) Compound light microscope

b) Reagents and glasswares

  • Bunsen flame
  • Wire loop
  • Clean grease-free slides
  • Marker pen
  • Crystal violet (Basic dye)
  • Gram’s iodine(mordant)
  • 95% ethanol (decolorizing agent)
  • 1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen (Overnight culture of Staphylococcus aureus on Blood Agar was taken for Gram staining.)

Preparation of Staphylococcus aureus smear

  • Take a clean, and grease-free slide for making a smear.
  • Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
  • With a straight wire touch the surface of a well-isolated colony from the blood agar and emulsify in the saline drop forming a thin film.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.


  1. Cover the smear with crystal violet and allow it to stand for one minute.
  2. Rinse the smear gently under tap water.
  3. Cover the smear with Gram’s iodine and allow it to stand for one minute.
  4. Rinse smear again gently under tap water.
  5. Decolorize the smear with 95% alcohol.
  6. Rinse the smear again gently under tap water.
  7. Cover the smear again gently with safranin for one minute.
  8. Rinse the smear again gently under tap water and air dry it.
  9. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.


Positive Control:   violet color, round in shape in single, pairs and cluster

Test: Purple color, round in shape and arrangement in singles, pairs, and cluster

Negative Control: red in color and rod in shape

Result and Interpretation

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-positive cocci in single, pairs, and cluster

Negative Control(NC): Gram-negative bacilli as shown above image.

Further Readings

  1. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  2. A handbook of Clinical Microbiology by Prof. Dr. Bharat Mani Pokhrel
  3.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  4.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  5. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  6. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
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