Gram staining  is a special technique which is used to stain bacteria. This technique was developed by Christian Gram in 1884. The stain  stain used in Gram staining is called Gram stain. Chemically Gram stain is a weakly alkaline solution of crystal violet or gentian violet.

On the basis of cell wall structure and its staining ability with Gram stain, bacteria are grouped into two categories. They are Gram positive bacteria and Gram negative bacteria
Cell wall of Gram positive bacteria
Cell wall of Gram negative bacteria
Cell wall is single layered and primarily made up of peptidoglycan
Cell wall is double layered and with an outer membrane outside to Peptidoglycan layer
Gram positive cell wall retain the primary stain of Gram staining (crystal violet) and appear purple after alcohol treatment
Gram positive cell wall lose the primary stain of Gram staining (crystal violet) after alcohol treatment; and appear pink with counter stain (safranin)
Thick peptidoglycan layer of about 20 to 80 nm
Peptidoglycan layer thin and single layered (about 5 to 10 nm thick)
Periplasmic space is small, if present
Large Periplasmic space* is present
Outer membrane is absent
Outer membrane is present outside to Peptidoglycan layer (about 7.5 to 10 nm thick)
Teichoic acid is present
Generally Teichoic acid is absent
Generally **Porins are absent
Porins are present


Very low lipid content (2-5%)
Very high lipid content (15-20%)
Generally Lipopolysaccharide (LPS) is absent**
Lipopolysaccharide (LPS) is present
Lysozyme degrade peptidoglycan wall cause lysis of cell (sensitive to lysozyme)
Lysozyme cannot degrade Gram negative bacteria as Peptidoglycan is protected by outer membrane
(resistant to lysozyme)
Example of Gram Negative Bacteria

* The region between cytoplasmic and outer membrane of the Gram-negative bacteria is called the periplasmic space
*Porins produce large, open, water-filled channels that allow the influx of normal, hydrophilic nutrient molecules and the efflux of waste products but exclude many antibiotics and inhibitors
 **only one Gram-positive organism, Listeria monocytogenes, has been found to contain LPS

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