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 a 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 and 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 a tichoic acid which is embedded within the peptidoglycan layers tychoic 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 pepticoglycan 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 wall 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 the 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 a 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 pepto-glycan 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 pepto-glycan layer shrinking and tightening it the large crystal violet iodine complex is not able to penetrate this titan peptoglycan layer and is thus trapped in the cell in gram-positive bacteria conversely the outer membrane of the gram-negative bacteria is degraded and the thinner peptoglycan layer of gram-negative cells is unable to retain the crystal violet iodine complex and color is lost and lastly a counter stain such as the weekly water soluble safranin is added to the sample because gram-negative bacteria are colorless after the alcohol wash the addition of the 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 the gram-positive cell as a result gram-positive bacteria are stained purple and gram-negative bacteria are stained pink or red do not forget to like the video and subscribe to our youtube channel