There are several factors that could result in a Gram-positive organism staining Gram-negatively:ġ. It is important to note that Gram-positivity (the ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon but a matter of degree. The few layers of peptidoglycan are unable to retain the crystal violet-iodine complex and the cell is decolorized. In the case of Gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. With the current theory behind Gram staining, it is thought that in Gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. © Daniel Cavanaugh, Mark Keen, authors, Licensed for use, ASM MicrobeLibrary. The Gram staining procedure involves four basic steps:įlash animation illustrating the interaction of the Gram's stain reagents at a molecular level The Gram-Negative Cell Wall Unit 1, Section IIB2b.The Gram-Positive Cell Wall Unit 1, Section IIB2a.The Prokaryotic Cell Wall Unit 1, Section IIB2.The outer membrane also contains a number of proteins that differ with the strain and species of the bacterium.įor further information on the Gram-negative and Gram-positive cell wall, see the following Learning Objects in your Lecture Guide: The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A embedded in the membrane and a polysaccharide portion extending outward from the bacterial surface. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. Only 10% - 20% of the Gram-negative cell wall is peptidoglycan. The Gram-negative cell wall, on the other hand, contains only 2-3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins (Figures 2A and 2B). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium. Some have a lipid attached (lipoteichoic acid). Teichoic acids, which extend through and beyond the rest of the cell wall, are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Interwoven in the cell wall of Gram-positive are teichoic acids. Chemically, 60 to 90% of the Gram-positive cell wall is peptidoglycan. In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20-80 nm thick and consisting of numerous interconnecting layers of peptidoglycan (Figures 1A and 1B). The material in the bacterial cell wall which confers rigidity is peptidoglycan. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. ![]() The terms positive and negative have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. Bacteria that stain purple with the Gram staining procedure are termed Gram-positive those that stain pink are said to be Gram-negative. ![]() It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. The Gram stain is the most widely used staining procedure in bacteriology.
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