![]() The alcohol increases the porosity of the cell wall by dissolving the lipids in the outer layers of the bacterium. Decolorization is the most important step! Decolorization serves as a protein-dehydrating reagent and a lipid solvent. The dye-mordant complex will not be removed from Gram-positive bacteria but is leached from Gram-negative cells during the alcohol or acetone (95% ethyl alcohol) in the Decolorization step. At this point, the cell types will both appear purple. In the second step of the procedure, Gram's Iodine is added to the smear as a mordant to complex with the crystal violet and forms an insoluble complex in Gram-positive cells. Initially, both Gram-positive AND Gram-negative cells are stained by the Primary Stain, the Crystal Violet. Since the best Gram-stained preparations are those made with fresh cultures (24 hours old may appear to be Gram-variable, in that some cells will appear purple, while others will appear red. Christian Gram, it separates the bacterial cells into two major groups: Gram-positive and Gram-negative, making it a critical tool in the classification and differentiation of microbes. Thanks to the Gram-stain, the most important differential stain used in Bacteriology, cell types or structures and their arrangements can be distinguished from each other on the basis of the stain that has been retained. If the primary stain is not removed or washed out during decolorization, the counterstain cannot be absorbed and the cell will retain the color of the primary stain (purple-blue). After decolorization, if the primary stain is removed, the decolorized cell will accept and assume the counterstain contrasting color (pink-red). Finally, the counterstain is applied ( Safranin), which contrasts in color to the primary stain. This is a protein-dehydrating agent and a lipid solvent. In order to establish a color contrast, this is followed by the addition of an acid/alcohol decolorizing reagent (ethyl alcohol, 95%), which may or may not remove the primary stain from the entire cell or just from some of the cell structures. This is followed by a second reagent ( Gram's Iodine), which acts as a mordant and helps the color to stick to the cell wall. The first reagent ( Crystal Violet) is the primary stain. The Gram-stain is a differential stain that requires the use of at least three chemical reagents applied sequentially to a heat-fixed smear. ![]() ![]() Microbiology History, Background, Introduction The Kirby Bauer Method and E Test for Antimicrobial Susceptibility Testing ![]() Streaking Agar Plates: 4 Quadrant Streak Methodīacterial Growth Patterns and Colony Types Diseases and Isolation of the Microbes that Cause Them ![]()
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