Gram staining

Gram staining

  • Gram staining is a method used to differentiate two groups of bacteria.
  • Based on the cell wall.
  • Developed by Hans Christian Gram in the 19th
  • This method is used to identify gram +ve gram –ve
  • After performing staining the Gram-positive bacterium stain violet and Gram-negative bacterium stain pink.
  • This technique of staining can’t define bacteria, used to general identification such as morphology.


How does Gram staining work?

  • Gram staining is a three-step process:

Staining with crystal violet dye, decolorization, counterstaining

  • Due to the difference of (thick and thin layer) cell wall, the cells are stained violet and red.
  • The cell wall of the bacterium is made up of peptidoglycan.

Steps of Gram staining:

  • Stain the cell using crystal violet dye.
  • Add Gram’s iodine. (to form a complex between dye and iodine CV-I complex)
  • Add decolorizer (ethyl alcohol/acetone).
  • Adding decolorizer dehydrates the cell.
  • After adding decolorizer, the CV-I complex trapped between gram-positive cell walls because it is very thick, and the CV-I complex in the case of gram-negative is dissolved with water.
  • Add counterstain (safranin)
  • Adding safranin doesn’t disturb the gram-positive cell it only affects gram-negative cells and stain red.


Gram-positive bacteria:

  • Contain a single membrane (monoderm) which is surrounded by a thick layer of peptidoglycan.
  • Stain purple after gram staining.
  • Teichoic acids are present on the peptidoglycan.

Gram-negative bacteria:

  • A thin layer of peptidoglycan is present.
  • LPS present (lipopolysaccharide)1


Difference between Gram Positive and Gram Negative bacteria:

Property Gram-positive Gram-negative
  • Cell wall thickness
Around 40-80nmnm Thinner than gram-positive,30nm
  • Gram staining
Stain blue(violet/purple) color in gram staining Stain pink in gram staining
  • LPS (Lipopolysaccharide)
Absent present
  • Teichoic acid
Cell wall contains teichoic acid (-ve charged)  Teichoic acids are absent
  • Periplasmic space
Small and single Two periplasmic space are present
  • Lipid content
Low High (because of LPS)
  • PG (peptidoglycan layer)
Thick and multilayered Thin and single-layered
  • Porins
Absent present
  • Flagellar structure
2 rings 4 rings
  • Toxins
Exotoxin Endo or exotoxin
  • Pathogenicity
Some belong to the pathogenic group Most of them are pathogens
  • Outer membrane
Absent Present
  • Mesosome
More prominent Less prominent
  • Morphology
Cocci or rods (spore-forming) Rods (non-spore forming)
  • Drug resistance
More susceptible More resistant to drugs
  • Examples
Staphylococcus, streptococcus… Escherichia, salmonella…
  • Resistant to drying
High Sensitive/low



  • Peptidoglycan is also known as
  • PG is a polymer of sugars and amino acids.
  • PG forms a mesh-like structure outside the plasma membrane.
  • The sugar molecules (of Murein) of alternating residues NAM and NAG linked by β-1,4 (glycosidic bond)
  • NAM (N-acetylglucosamine), NAG (N-acetylmuramic acid).
  • NAM is a peptide chain of 3-5 AA (amino acids).
  • In the peptidoglycan of gram-positive L-Lys is present and in gram-negative in place of L-Lys DAP (diaminopimelic acid) is present.


Can we heat fix the cell in this method of staining? Explain why?

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