Gram stain is the most important staining procedure in Microbiology. Most bacteria can be broadly classified as Gram-positive or Gram-negative. Gram-positive bacteria stain purple, whereas gram-negative bacteria stain pink. This difference is due to the ability of gram-positive bacteria to retain primary stain (crystal violet) even after decolorization with acid-alcohol.
The major structural difference between Gram-positive and Gram-negative bacteria is the thickness of peptidoglycan and the presence of the outer membrane. Gram-negative bacteria have a thin peptidoglycan layer covered by an outer lipid-containing membrane (lipopolysaccharide layer), whereas Gram-positive bacteria have thick peptidoglycan and no outer membrane.
The Gram-Positive Cell Wall
The cell wall of gram-positive bacteria is much thicker than of Gram-negative bacteria and consists primarily of a single type of molecule.
Peptidoglycan Layer
The peptidoglycan layer is the outermost covering of the Gram-positive cell wall and constitutes as much as 90% of the Gram-positive cell wall. Gram-positive bacteria have several sheets of peptidoglycan stacked and cross-linked by glycan strands. Many gram-positive bacteria have teichoic acids (polymers of glycerol phosphate or ribitol phosphate) covalently bonded to muramic acid in the wall peptidoglycan or membrane lipids (lipoteichoic acids).
The Gram-Negative Cell Wall
The gram-negative cell wall is more chemically complex than the gram-positive and consists of at least two layers.
Lipopolysaccharide Layer
The outer membrane (lipopolysaccharide layer, LPS in short) is the outermost covering of the Gram-negative cell wall.
Peptidoglycan layer
Beneath the outer membrane in Gram-negative bacteria lies a thin sheet of peptidoglycan, which constitutes only 10% of the cell wall of Gram-negative. The outer membrane contains a lipid bilayer bonded with polysaccharides (lipopolysaccharide).
Periplasmic space
Periplasm is the area between the cytoplasmic membrane and the outer membrane in gram-negative Bacteria. Whether periplasmic space is present in gram-positive bacteria is the subject of debate. The periplasm contains many proteins, several of which function in transport and are called periplasmic binding proteins. This function is done by the ABC transport system in Gram-positive bacteria, as Gram-positive bacteria lack periplasmic space. Periplasm also contains several enzymes involved in the degradation of macromolecules and detoxifying environmental solutes, including antibiotics.
Differences between Gram-positive and Gram-negative bacteria
| Properties | Gram-Positive Bacteria | Gram-Negative Bacteria |
| Thickness of cell wall | Thicker than Gram-negative bacteria, around 20 to 25 nm | The cell wall of Gram-negative bacteria is generally thinner, 11 to 15 nm in diameter |
| Gram reaction | Gram-positive bacteria stain a deep blue color (violet/purple) in the Gram staining technique. | Gram-negative bacteria stain pink to red color in the Gram staining technique. |
| Lipopolysaccharide (LPS) layer | The lipopolysaccharide layer, also known as the outer membrane, is absent in Gram-positive bacteria. | LPS is only present in Gram-negative bacteria. |
| Peptidoglycan layer | A thick (multilayered) peptidoglycan layer is present in Gram-positive bacteria. It accounts for 50% or more of the dry weight of the wall of some Gram-positive bacteria. | Thin (single-layered). Around 10% weight of the cell wall of Gram-negative bacteria. |
| Teichoic acids | Cell wall of gram-positive bacteria contains teichoic acids. | Teichoic acid is absent in Gram-negative bacteria |
| Periplasmic space | The presence of periplasmic space Gram-positive bacteria is a subject of decade. Generally, it is regarded that periplasmic space is absent in Gram-positive bacteria. | There are two periplasmic spaces in Gram-negative bacteria; one between the murein and inner cell membrane and the other between the murein and outer cell membrane. |
| Flagellar structure | Two rings in the basal body | Four rings in the basal body |
| Toxins produced | Primarily exotoxins | Primarily endotoxins, the LPS layer has an endotoxic property. |
| Lipid content | Low | High around 11 to 22% of the dry weight of the cell wall (because of the lipid-rich LPS layer). |
| Action of Lysozyme | Cell wall of Gram-positive bacteria is easily destroyed by the action of lysozyme. After digestion of the Peptidoglycan layer, Gram-positive bacteria become protoplast. | Gram-negative bacteria are refractory to lysozyme because large protein molecules cannot penetrate the LPS layer. After digestion of the Peptidoglycan layer, Gram-negative bacteria become spheroplasts. |
| Examples | Clostridium spp, Bacillus spp, Listeria monocytogenes, Staphylococcus, Streptococcus, etc. | Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Vibrio cholerae. |
Spheroplasts: Gram-negative bacteria with the intact cytoplasmic membrane of the protoplast plus the outer membrane (LPS layer) of the cell wall, after the peptidoglycan layer is destroyed by lysozyme or its synthesis inhibited by antibiotics.
Protoplasts: Cells whose walls have been completely removed are incapable of normal growth and division.
