How to identify gram negative bacteria?


I am not asking about the general staining.

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Tina C. Spencer 1 year 1 Answer 297 views Regular Member 0

Answer ( 1 )

  1. Gram negative bacterial cell wall:

    Gram negative bacteria have different cell wall than gram positive bacteria. Because of the change in cell wall stricture, the bacteria are classified as gram positive bacteria and gram negative bacteria. Gram negative bacteria cell wall has some differences than the gram positive cell wall. They have single layer of peptidoglycan. The cell wall thickness is 70-120 Å. It is two layered cell wall that have 20-30% lipid content and is 10-20% murein content. There are three components in the gram negative bacteria cell wall.

    1. Peptidoglycan
    2. Lipopolysaccharides
    3. Teichoic acid


    1. Peptidoglycan:

    Peptidoglycan is the monomeric form of carbohydrate that is only found in bacteria only. They are the complex polysaccharides that synthesize the cell wall of bacteria. This peptidoglycan is the polymers of sugar and amino acids. The carbohydrates that are N-acetylglucosamine and N- acetylmuramic acid are linked with the amino acids. Three to five peptide chains linked the sugar and protein together. These amino acids are homopeptide and are similar to each other. These polymers combine to give the structure of bacterial cell wall. Gram negative bacteria have low content of peptidoglycan. They have the thin layer of peptidoglycan that is present in the cell wall. Gram positive bacteria have high content of peptidoglycan that is present in their cell wall.


    Peptidoglycan is present in bacterial cell wall as crystal lattice form. Two amino sugar chains are present which is called N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). These NAM and NAG units are linked with the three to five linker peptides. These units are cross linked with the amino acids residues. These cross linked amino acids are oligopeptide. Peptidoglycan monomers are synthesized in the cytosol of bacterial cell. These monomers are synthesized by the bacterial enzymes that are present in the cytoplasm. Bactoprenol are the transporter proteins that transport the monomers of peptidoglycan to the cell wall. These monomers arranged themselves in the cell wall and prepare the cell wall for bacteria.

    Inhibition of peptidoglycan:

    The synthesis of peptidoglycan is inhibited to stop the growth of bacteria. The bacterial infection stop and its inhibition give bactericidal effect to the organism. It is inhibited by various ways;

    • The peptidoglycan monomers synthesis is blocked by using antibiotics. If the monomers are not synthesized, the cell wall is not prepared and synthesized properly.
    • The cross linker peptide synthesis can also be prohibited to stop bacterial growth. If linker peptide is inhibited, the monomers are not linked and cell wall is not synthesized.
    • If the monomers are present in cytosol, the joining peptide that binds the monomers can be blocked.
    • The linker peptide can be altered by using identical precursor. These precursor unable the cell wall to bind and form cell wall.
    • Bactoprenol that transport the monomers is blocked by using antibiotics. The transportation of monomers is blocked and then cell wall synthesis is blocked.


    1. Lipopolysaccharides:

    The outer membrane is present in gram negative bacteria that surround the cell wall of bacteria.  This membrane is composed of lipo-protein, lipopolysaccharides and phospholipids. The lipopolysaccharide is composed of two portion i.e. polysaccharides and lipid A portion. Lipid A portion of this membrane is an endotoxin in nature. These endotoxin is the distinguish feature of gram negative bacteria. The lipid A portion induces fever and shock by lowering the blood pressure of whole body. Vasodilation of blood vessel occur when the whole BP of body become low. This can cause death of the organism.


    This membrane has many pores that are called as porins. Porins are permeable lipo-proteins and allow the transportation of substrate and nutrient to move in the cell. These porins can either be selective or non-selective. Selective porins allow the transportation of selective substances while non-selective porins allows any substance to move in the cell. The outer membrane has many structures that help the bacteria to attach to the surfaces.

    Periplasmic space;

    Periplasmic space is the area between the outer membrane and cell wall that is present in gram negative bacteria. This space plays a vital role for the bacteria as it stores macromolecules. Macromolecules help in the metabolism of cell and environmental response. It contains many binding proteins that bind amino acids, vitamins, iron and enzymes. These substances are essential for the nutrition of bacteria.

    1. Teichoic acid:

    Teichoic acid is present in the gram negative bacteria cell wall. It is present in minute quantity and present as lipo-teichoic acid. These are polymers of polyglycerol that are supplemented with phosphates and some sugar residues. Their function is unknown up till now. Some theory revealed that they are essential for the assembly of peptidoglycans monomers in the cell wall.


    Examples of gram negative bacteria:

    • Escherichia coli
    • Salmonella
    • Shigella
    • Proteus
    • Vibrio
    • Campylobacter

    Staining of gram negative cell wall:

    Gram negative bacterial cell wall has less component of peptidoglycan. This low content of peptidoglycan doesn’t retain the primary dye color. Primary stain and mordant bind with the peptidoglycan content of cell wall and make a very weak bonding as CV-PG-I complex. They have high content of lipid content in the cell wall. This CV-PG-I complex and lipids of the cell wall is washed by using alcohol. Alcohol dissolves the lipid content of cell wall and thus causes empty spaces between the cell wall. Saffarine enters in the empty spaces of the cell wall and bind with them. The secondary dye retains in the empty space and thus, don’t wash away. Then, during microscopy, these bacteria appear as pink color under the microscope.

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