![]() ![]() It is either acetone or ethanol (95%) or a mixture of acetone and ethanol in ratio 1:1 by volume. It interacts with CV+ and forms a CVI complex which gets trapped in the dehydrated peptidoglycan layer of the Gram-Positive cell wall. It is an aqueous solution of iodine and potassium iodide used as mordant in Gram staining. It provides violet color to Gram-Positive bacteria. ![]() In Gram Staining, it is used as a basic dye in the ionized form of CV+ and Cl. It also shows antibacterial and antifungal properties, hence used in sterilization and disinfection. In microbiology and molecular biology, it is used for staining bacteria, histological slide staining, DNA staining, etc. It is used for staining textiles, papers, and fibers, in ball pens, and chemicals like detergents, fertilizers, etc. Its color depends on the pH of the dissolving medium such as, at pH -1.0 or below, it appears yellow, and at acidic pH of 1 to 2 it appears green, at neutral pH, it appears purple (deep blue-violet), and at highly basic pH it appears colorless. It is also known as hexamethyl pararosaniline chloride or methyl violet 10B or gentian violet. It is an intensely purple-colored organic compound chemically called triphenylmethane dye. Gram staining procedure uses different chemicals and dyes that can be grouped 1. Gram-positive and gram-negative cell wall structure. Hence, safranin can’t stain them red or pink and Gram-Positive bacteria reveal the purple or violet color. Whereas, there is no space to enter inside the dehydrated Gram-Positive cell wall due to CVI complex and dehydration. Stain precipitate free#When counterstain, positively charged safranin, is added, it interacts with the free negatively charged components in Gram-Negative cell wall and membrane and bacteria becomes pink/red. So, the decolorizing solution dehydrates the peptidoglycan layer trapping all the CVI complexes inside the cell wall and bacteria retain the purple or violet color of crystal violet. Whereas in Gram-Positive bacteria, there is no outer membrane, and the peptidoglycan layer is also thick with higher cross-linkage. This causes cells to lose most of the CVI complexes. The peptidoglycan layer is thin with less cross-linking in the Gram-Negative cell wall, hence becoming leaky. The outer membrane of the Gram-Negative bacterial cell wall is dissolved exposing the peptidoglycan layer. When decolorizing solution (ethanol or a mixture of ethanol and acetone) is added it interacts with lipids in the cell wall. When Gram’s Iodine is added as mordant, the iodine (I – or I -3 ion) interacts with CV + ion and forms CV-I complex within cytoplasm and cell membrane and cell wall layers. The CV + ion interacts with negatively charged components of the cell wall. These ions easily penetrate the cell wall components of both positive and negative bacteria. In an aqueous solution of crystal violet dye, their molecules dissociate into CV + and Cl – ions. Bacteria having a thin peptidoglycan layer with lesser cross-linkage lose primary stain during decolorizing and gain counter stain appearing pink or red. Bacteria having cell walls with a thick layer of peptidoglycan will resist decolorization of primary stain and appear violet or purple. ![]() Gram staining and differentiation are based on the differences in cell wall structure and composition of bacteria. Using this staining technique, bacteria can be differentiated into two groups hence it is called the differential staining technique. These bacteria are called Gram-Negative bacteria. ![]() The other group of bacteria with Gram-Negative cell wall will lose primary stain and take up the counterstain and appears pink or red under the microscope. These bacteria are termed Gram-Positive bacteria. Those bacteria with Gram-positive cell walls will retain primary stain and appear violet or purple. This staining technique uses two stains crystal violet as primary stain and safranine as a counterstain. It is generally the first test performed on bacteria during their identification and observation process. It is the most widely used and the most important staining technique in bacteriology, especially in medical bacteriology. Gram staining is a differential bacterial staining technique used to differentiate bacteria into Gram Positive and Gram Negative types according to their cell wall composition. ![]()
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