![]() ![]() ![]() Before starting the whole process, a smear is needed to be prepared with the help of a loop. The overall procedure of the Gram stain is completed in four steps. Gram-positive cells retain the purple color of the primary stain.įigure 2: Steps involved in Gram staining Gram-negative cells undergo decolorization, so only they absorb the counterstain and become pink. Finally, the final reagent safranin is used as a counterstain to stain only the colorless or unstained cells. Gram-negative bacterial contain higher lipid concentration in the outer layer of cells, do not close appreciably on dehydration of cell wall protein, results in the release of CV-I complex, and cells become unstained. The lower amount of lipid is readily dissolved by alcohol treatment, forms minute cell wall pores, and is then closed by dehydrating effect of alcohol. Gram-positive cells have low lipid concentration, which is essential for the retention of the Mg-RNA-CV-I complex. In the third step of the Gram staining procedure, ethyl alcohol (95%) is used as a decolorizing agent that functions as a lipid solvent and as a protein dehydrating agent. The complex is more difficult to remove than the CV-I complex of Gram-negative cells. Only in Gram-positive cells, the CV-I complex binds to the magnesium ribonucleic acid component of the cell wall and form the magnesium ribonucleic acid-crystal violet-iodine complex (Mg-RNA-CV-I). It binds to the primary stain to form an insoluble crystal violet-iodine complex (CV-I), and all cells will appear purple-black. After that, Gram’s iodine is used, which function as a mordant. Next, Crystal violet dye is used, and it stains all the cells violet. For Gram staining, at first heat-fixed smear is prepared with a fresh bacterial culture. Crystal violet is used as a primary stain, gram’s iodine is a mordant, ethyl alcohol is a decolorizing agent, and, finally, safranin as a counterstain. įour chemical reagents are essential for Gram staining. Finally, the counterstain is used that has a contrasting color to that of the primary stain. After that decolorizing agent is used to create color contrast based on the chemical constituents of the bacterial cell wall, the decolorizing agent may or may not remove primary stain from entire cells or specific cell structures. ![]() First, the primary stain is used, and its function is to impart its color to all cells. So, the main reason for showing different colors is the thick peptidoglycan layer which retains the color of crystal violet for Gram-positive and on the other hand, the thin peptidoglycan layer is responsible for stacking the primary stain in between the inner and outer membrane of the Gram-negative bacteria and for this, they show the color of counterstain which is pink.Īt least three chemical reagents are applied sequentially to a heat-fixed smear in a differential straining procedure. Compared with Gram-positive bacteria, Gram-negative bacteria possess a thin peptidoglycan layer with an absence of teichoic acid, which helps to stain the color of the counterstain in pink. The Gram-positive bacteria show purple color after the observation under the microscope. The cell wall of Gram-positive bacteria contains a thick peptidoglycan layer with teichoic acid, which retains the color of crystal violet. The key difference between Gram-positive and Gram-negative bacteria lies in their cell wall structure composition. Gram-negative bacteria retain the color of safranin or counterstain and show pink color. It is already known that Gram-positive bacteria retain the color of crystal violet or the color of the primary stain and show the purple color. įigure 1: Cell wall of Gram-positive Vs Gram-negative bacteria. Gram stain is an essential tool for the differentiation and classification of microorganisms. The cell wall of gram-positive bacteria retain the CV-I complex after treatment with ethyl alcohol and appear purple, but gram-negative bacteria decolorize following such treatment and appear pink. It is used to distinguish between Gram-positive and Gram-negative bacteria based on differential staining with a crystal violet-iodine complex (CV-I) and a safranin counterstain. The role of the Gram staining procedure is crucial in bacteriology. Gram stain is a well-known differential staining procedure, and it was developed in 1884 by the Danish bacteriologist Hans Christian Joachim Gram. ![]()
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