<P> Gram - positive bacteria have a thick mesh - like cell wall made of peptidoglycan (50--90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram - negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin . There are four basic steps of the Gram stain: </P> <Ul> <Li> Applying a primary stain (crystal violet) to a heat - fixed smear of a bacterial culture . Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure </Li> <Li> The addition of iodide, which binds to crystal violet and traps it in the cell </Li> <Li> Rapid decolorization with ethanol or acetone </Li> <Li> Counterstaining with safranin . Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain . </Li> </Ul> <Li> Applying a primary stain (crystal violet) to a heat - fixed smear of a bacterial culture . Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure </Li> <Li> The addition of iodide, which binds to crystal violet and traps it in the cell </Li>

What is the primary stain in the gram stain