The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification. The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained. With the dissolution of the lipid layer, gram negatives lose the primary stain. Initially, all bacteria take up crystal violet dye however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Subsequently, a decolorizer, often solvent of ethanol and acetone, is used to remove the dye. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.

The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia. The Gram staining is one of the most crucial staining techniques in microbiology.

Gram negative and gram positive