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What is the most critical step in Gram staining?

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The decolorizing step is the most critical in Gram staining. The outcome in this step is based on how easily the cell releases the crystal violet-iodine complex.

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Discovered by Dr. Hans Christian Gram in 1884, the Gram stain is a differential stain that is used in identifying bacteria. Dividing bacterial cells in two major groups (Gram positive and Gram negative), it is a critical tool when classifying and differentiating bacteria. The two stains required are the primary and counterstain. Over-decolorizing causes the primary stain to disappear, and Gram positive appears Gram negative. On the other hand, under-decolorizing does not remove the crystal violent-iodine complex, which causes Gram negative bacteria to appear positive.

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