Bacterial cell wall is found outside of the cell membrane. It’s an additional layer that typically provides some strength that the cell membrane lacks.It helps maintain the cell shape and it also protects the cell from osmotic lysis, as the cell moves from one environment to another. The bacterial cell wall owes its strength to a layer composed of a substance referred to as peptidoglycan. Peptidoglycan is a polymer made up of sugars and amino acids which forms a kind of mesh.

However, the structure of the cell wall in bacteria is not the same in all bacteria. And this causes bacteria to be divided into two separate groups as gram-positive bacteria and gram-negative bacteria.

In gram-positive bacteria, the cell wall consists of many layers of peptidoglycan, forming a thick, rigid structure.The additional component in a gram positive cell wall is teichoic acid which is embedded within the peptidoglycan layers. Teichoic acid contributes to the overall rigidity of the cell wall, which is important for the maintenance of the cell shape.

In contrast the cell wall of gram-negative bacteria comprises two layers: the inner peptidoglycan layer, which is much thinner than in gram positive cell walls, and an additional outer membrane unique to the gram negative cell wall.The outer membrane is composed of a lipid bilayer, very similar in composition to the cell membrane. It differs from the cell membrane by the presence of large molecules known as lipopolysaccharide or LPS , which are anchored into the outer membrane and project from the cell into the environment. LPS is made up of three different components first one is the O antigen which represents the outermost part of the structure and the second one is  core polysaccharide, and the last one is lipid A, which anchors the LPS into the outer membrane. LPS is known to serve many different functions for the cell, such as helping to stabilize the outer membrane, and providing protection from certain chemical substances by physically blocking access to other parts of the cell wall. In addition, LPS plays a role in the host response to pathogenic gram negative bacteria. The O-antigen triggers an immune response in an infected host, causing the generation of antibodies specific to that part of LPS. And Lipid A acts as a toxin, specifically an endotoxin, causing general symptoms of illness such as fever and diarrhea.


So how can the gram staining method separate these two types of bacteria?


The mechanism of the Gram stain is based on differences in the cell wall structure of gram-positive and gram-negative bacteria. First cells are stained with crystal violet dye. Crystal violet, the primary stain, stains both gram-positive and gram-negative cells purple because the dye combines with the peptidoglycan. Next, iodine solution is added to form a complex between the crystal violet and iodine. When iodine is applied, it forms large crystals with the crystal violet dye that are not soluble in water. Then a decolorizer such as alcohol is added to the sample which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.
And lastly a counterstain, such as the weakly water soluble safranin, is added to the sample.Because gram-negative bacteria are colorless after the alcohol wash, the addition of safranin turns the cells pink or red. Although gram-positive and gram-negative cells both absorb safranin, the pink or red color of safranin is masked by the darker purple dye previously absorbed by gram-positive cell.
As a result, gram-positive bacteria are stained purple and gram-negative bacteria are stained red or pink.

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