What Is the Most Critical Step in Gram Staining?

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.