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.
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.